Compositions and methods useful for the diagnosis and treatment of heparin induced thrombocytopenia/thrombosis

ABSTRACT

The invention includes compositions, kits and methods comprising a monoclonal antibody which shares key functional properties with the polyclonal antibodies which participate in the pathogenesis of heparin induced thrombocytopenia/thrombosis (HIT/HITT) in a mammal. The monoclonal antibody of the invention preferentially binds with a PF4/heparin complex relative to the binding of the antibody with PF4 or heparin alone. The monoclonal antibody of the invention also binds specifically with PF4 in a complex with other glycosaminoglycans besides heparin, and also activates platelets. The monoclonal antibody of the invention is useful in methods for diagnosing and treating HIT/HITT in a mammal. A humanized version of the monoclonal antibody of the invention is also included, along with a process for humanizing the monoclonal antibody of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/615,872, filed Jul. 13, 2000, which claims the benefit of U.S.provisional patent application 60/143,536, filed on Jul. 13, 1999.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH AND DEVELOPMENT

This invention was supported in part by U.S. Government funds (NHLBI/NIHGrant No. 2RO1 HL 35246, NIH/NCI Grant No. P20RR11830 and NIH Grant No.KO8 HL04009), and the U.S. Government may therefore have certain rightsin the invention.

BACKGROUND OF THE INVENTION

Heparin-induced thrombocytopenia (HIT) is a drug-induced blood disorderthat affects 1-5% of patients who are treated with the blood-thinningdrug heparin (Chong, 1995, British Journal of Haematology, 89(3):431-9).Patients who develop HIT are at risk for developing life-threateningclots in the heart, the brain, the extremities or the lungs (Arepally etal., 1998, Clinical Reviews in Allergy and Immunology, 16:1-11). HIT iscaused by antibodies which bind specifically with a complex comprisingthe protein Platelet Factor 4 (PF4) and heparin, a sugar compoundclassified as a glycosaminoglycan (Amiral et al., 1992, Thrombosis &Haemostasis, 68(1):95-6). Antibodies directed against the PF4/heparincomplex can be demonstrated in the blood of patients with HIT, and areassociated with the clotting problems seen in these patients. Antibodiesto the PF4/heparin complex which have been isolated from humans (Suh etal. 1998, Blood, 91(3):916-22) and mice (Blank et al., 1997, Clin. Exp.Immunol. 108(2):333-9) are polyclonal and heterogeneous since they arederived from the natural polyclonal immune response of an organism to anantigen.

Although autoantibodies against the PF4/heparin complex can beidentified in the plasma of more than 90% of patients who develop theclinical syndrome of HIT (Arepally et al., 1995, Am. J. Clin. Pathol.104:648), these antibodies can also be demonstrated in a significantproportion (15-70%) of asymptomatic patients repetitively exposed toheparin (Amiral et al., 1996, Am. J. Hematol. 52:90; Visentin et al.,1996, J. Lab. Clin. Med. 128:376; Trossaert et al., 1998, Br. J.Haematol. 101:653; Bauer et al., 1997, Circulation 95:1242). The reasonwhy only a subset of immunized patients develop symptomatic disease isunknown (Schmitt et al., 1993. Am. J. Med. Sci. 305:208; Warkentin etal., 1996, Am. J. Med. 101:502; Sheridan et al., 1986, Blood 67:27). Themolecular basis for a pathogenic role of antibodies to the PF4/heparincomplex has been difficult to establish, since the antibodies arepolyclonal and polyspecific.

Heterogeneity in disease expression may, in part, reflect differences inco-morbid factors that predispose to thrombosis, such asatherosclerosis, surgery and vascular trauma (Boshkov et al., 1993, Br.J. Haematol. 84:322; Lee et al., 1998, Thromb. Haemost. 79:50). Othershave implicated differences in antibody titer (Suh et al., 1997, Am. J.Hematol. 54:196), affinity (Suh et al., 1998, Blood 91:916), isotype(Amiral et al., 1996, Br. J. Haematol. 92:954), subclass (Suh et al.,1997, Am. J. Hematol. 54:196; Arepally et al., 1997, Blood 89:370), andplatelet Fc receptor polymorphism (FcγRIIA-H/R¹³¹) (Carlsson et al.,1998, Blood 92:1526) in affected individuals. However, it is clear thatsuch serologic or clinical differences do not permit unambiguoussegregation of asymptomatic patients with anti-PF4/heparin complexantibodies from those who develop thrombocytopenia and those who developthrombocytopenia and thrombosis (Arepally et al., 1997, Blood 89:370;Bachelot-Loza et al., 1998, Thromb. Haemost. 79:523; Herbert et al.,1998, Thromb. Haemost. 80:326).

An additional explanation for the observed heterogeneity in diseaseexpression may lie in the heterogeneity of anti-PF4/heparin complexantibodies themselves. Because PF4 modulates heparin-dependentanti-thrombin (Stern et al., 1985, J. Clin. Invest. 75:272) and proteinC co-factor activities (i.e., pro- and anti-coagulant activities {Dudeket al., 1997, C. J. Biol Chem 272:31785}), the effects on these or othercoagulant functions of PF4 by antibodies to the PF4/heparin complexremains to be elucidated. Anti-PF4/heparin complex antibodies differ intheir antigen specificities, although the responsible determinants havenot been clearly delineated (Suh et al., 1997, Am. J. Hematol. 54:196;Ziporen et al., 1998, Blood 92: 3250; Amiral et al., 1996, Blood 88:410;Horsewood et al., 1996, Br. J. Haematol. 95:161). However, thepolyclonal nature of the naturally occurring immune response complicatesany attempt to determine whether a subset of anti-PF4 antibodies areresponsible for thrombosis.

Because of the limited and heterogeneous supply of antibodies frompatients with heparin induced thrombocytopenia and/or thrombosis(HIT/HITT), it has been difficult to characterize the pathogenicproperties of antibodies to the PF4/heparin complex. Furthermore, indiagnosing HIT/HITT, antibodies to the PF4/heparin complex are needed asa positive control. Until now, tests have relied on the availability ofantibodies from patients with HIT/HITT as a “positive control”. Thus,there remains a need in the art for compositions and methods which areuseful in the diagnosis and treatment of HIT/HITT, or in the elucidationof the role of antibodies to the PF4/heparin complex in the developmentof HIT/HITT. The present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a composition comprising a monoclonal antibodywhich is capable of binding specifically with a PF4/heparin complex,wherein the antibody preferentially binds with the PF4/heparin complexrelative to the binding of the antibody with either PF4 or heparinalone.

In one embodiment, the monoclonal antibody is capable of bindingspecifically with a glycosaminoglycan which is not heparin.

In another embodiment, the monoclonal antibody is capable of activatingplatelets in the presence of PF4 and heparin.

In one aspect, the antibody comprises a heavy chain polypeptide havingan amino acid sequence which shares at least about 80% homology with SEQID NO:1 and a light chain polypeptide having an amino acid sequencewhich shares at least about 80% homology with SEQ ID NO:2.

In another aspect, the antibody is a murine monoclonal antibody (KKO)which comprises the heavy chain polypeptide of SEQ ID NO:1 and the lightchain polypeptide of SEQ ID NO:2.

In one embodiment, the antibody is a humanized antibody.

The invention also includes a composition comprising an isolated nucleicacid, wherein the isolated nucleic acid encodes an antibody which iscapable of binding specifically with a PF4/heparin complex, wherein theantibody preferentially binds with the PF4/heparin complex relative tothe binding of the antibody with either PF4 or heparin alone theisolated nucleic acid comprising a nucleotide sequence which shares atleast about 80% homology with SEQ ID NO:3 and a nucleotide sequencewhich shares at least about 80% homology with SEQ ID NO:4.

In one embodiment, the isolated nucleic acid comprises the nucleotidesequences of SEQ ID NO:3 and SEQ ID NO:4.

In another embodiment, the isolated nucleic acid encodes a humanizedmonoclonal antibody.

In one aspect, the composition is in the form of a pharmaceuticalcomposition.

The invention also includes a method of making a humanized monoclonalantibody which is capable of binding specifically with a PF4/heparincomplex, wherein the antibody preferentially binds with the PF4/heparincomplex relative to the binding of the antibody with either PF4 orheparin alone. The method comprises a) obtaining a monoclonal antibodywhich is capable of binding specifically with a PF4/heparin complex,wherein the antibody preferentially binds with the PF4/heparin complexrelative to the binding of the antibody with either PF4 or heparinalone; b) humanizing the antibody in a), whereby a humanized monoclonalantibody is made.

In one aspect, the monoclonal antibody in a) comprises a heavy chainpolypeptide having an amino acid sequence which shares at least about80% homology with SEQ ID NO:1 and a light chain polypeptide having anamino acid sequence which shares at least about 80% homology with SEQ IDNO:2.

In another aspect, the monoclonal antibody in a) is a murine monoclonalantibody (KKO) which comprises the heavy chain polypeptide of SEQ IDNO:1 and the light chain polypeptide of SEQ ID NO:2.

The invention also includes a method of diagnosing HIT/HITT in a mammal.The method comprises a) assessing the level of a polyclonal antibody ina sample of a bodily fluid or tissue obtained from the mammal, whereinthe polyclonal antibody binds specifically with a PF4/heparin complexand preferentially binds with the PF4/heparin complex relative to thebinding of the polyclonal antibody with either PF4 or heparin alone; b)comparing the level of the polyclonal antibody in the sample with thelevel of a monoclonal antibody which is specific for the PF4/heparincomplex in a positive control sample for HIT/HITT, wherein themonoclonal antibody preferentially binds with the PF4/heparin complexrelative to the binding of the monoclonal antibody with either PF4 orheparin alone; and c) determining from a) and b) whether the level ofthe polyclonal antibody in the sample is statistically similar to thelevel of the monoclonal antibody in the positive control sample, whereinwhen the level of the polyclonal antibody is statistically similar tothe level of the monoclonal antibody in the positive control sample,then HIT/HITT is diagnosed in the mammal.

In one aspect, the mammal is a human.

In another aspect, the monoclonal antibody is a murine monoclonalantibody comprising a heavy chain polypeptide of SEQ ID NO:1 and a lightchain polypeptide of SEQ ID NO:2.

In one embodiment, the level of the polyclonal antibody and the level ofthe monoclonal antibody are each assessed using an assay independentlyselected from the group consisting of an ELISA assay, a Western blottingassay, a serotonin release assay, a platelet aggregation assay, alumi-aggregometry assay and a flow cytometry assay.

The invention also includes a method of assessing the level of apolyclonal antibody in a bodily fluid or tissue sample obtained from amammal, wherein the polyclonal antibody binds specifically with aPF4/heparin complex and preferentially binds with the PF4/heparincomplex relative to the binding of the polyclonal antibody with eitherPF4 or heparin alone. The method comprises a) assessing the level of thepolyclonal antibody in the bodily fluid or tissue sample obtained fromthe mammal; b) comparing the level of the polyclonal antibody in thesample with the level of a monoclonal antibody which is specific for thePF4/heparin complex in a reference standard which comprises themonoclonal antibody, wherein the monoclonal antibody preferentiallybinds with the PF4/heparin complex relative to the binding of themonoclonal antibody with either PF4 or heparin alone; and c) determiningfrom a) and b) the level of the polyclonal antibody in the sample,whereby the level of the polyclonal antibody in the sample is assessed.

The invention also includes a method of identifying a functional elementof an antibody, wherein the functional element participates in thepathogenesis of HIT/HITT in a mammal. The method comprises a) preparingone or more deletion or substitution mutants of a monoclonal antibodywhich bind specifically with a PF4/heparin complex and whichpreferentially bind with the PF4/heparin complex relative to the bindingof either PF4 or heparin alone, wherein the one or more deletion orsubstitution mutants lack a portion of the amino acid sequence of theFab region of the monoclonal antibody; b) assessing the ability of eachof the deletion or substitution mutants to bind specifically with aPF4/heparin complex and to preferentially bind with the PF4/heparincomplex relative to the binding of the deletion or substitution mutantwith either PF4 or heparin alone; c) identifying, from b) one or more ofthe deletion or substitution mutants which does not preferentially bindwith the PF4/heparin complex relative to the binding of the deletion orsubstitution mutant with either PF4 or heparin alone; and d) determiningfrom c) and a) the corresponding deleted portion of the amino acidsequence of the monoclonal antibody which participates in thepreferential binding with the PF4/heparin complex, whereby a functionalelement of the monoclonal antibody which participates in thepathogenesis of HIT/HITT is identified.

In one aspect, the monoclonal antibody comprises a heavy chainpolypeptide having an amino acid sequence which shares at least about80% homology with SEQ ID NO:1 and a light chain polypeptide having anamino acid sequence which shares at least about 80% homology with SEQ IDNO:2.

The invention also includes a method of treating HIT/HITT in a mammal.The method comprises a) administering to the mammal a compositioncomprising a monoclonal antibody or a functional element thereof whichbinds specifically with a PF4/heparin complex and which preferentiallybinds with the PF4/heparin complex relative to the binding of themonoclonal antibody with either PF4 or heparin alone, wherein themonoclonal antibody or the functional element thereof is present in thecomposition in an amount effective to competitively inhibit the specificbinding of a polyclonal antibody in the mammal to the PF4/heparincomplex; and b) inhibiting the specific binding of the polyclonalantibody in the mammal with the PF4/heparin complex, thereby treatingHIT/HITT in the mammal.

In one aspect, the mammal is a human.

In another aspect, the monoclonal antibody comprises a heavy chainpolypeptide having an amino acid sequence which shares at least about80% homology with SEQ ID NO:1 and a light chain polypeptide having anamino acid sequence which shares at least about 80% homology with SEQ IDNO:2.

In a further aspect, the monoclonal antibody is a humanized antibody

In one embodiment, the monoclonal antibody is a murine monoclonalantibody comprising a heavy chain polypeptide having SEQ ID NO:1 and alight chain polypeptide having SEQ ID NO:2.

The invention also includes a method of identifying a compound which isa modulator of the specific binding of an antibody to a PF4/heparincomplex. The method comprises a) contacting the compound with theantibody and the PF4/heparin complex; and b) assessing the effect of thecompound upon the specific binding of the antibody to the PF4/heparincomplex, wherein a higher or lower level of specific binding of theantibody to the PF4/heparin complex in the presence of the compoundcompared with the level of specific binding of the antibody to thePF4/heparin complex in the absence of the compound is an indication thatthe compound is a modulator of the specific binding of an antibody to aPF4/heparin complex.

In one aspect, the antibody is a monoclonal antibody.

In another aspect, the antibody is a polyclonal antibody.

In one embodiment, the antibody is a murine monoclonal antibodycomprising a heavy chain polypeptide which shares at least about 80%homology with SEQ ID NO:1 and a light chain polypeptide which shares atleast about 80% homology with SEQ ID NO:2.

In another embodiment, the antibody is a murine monoclonal antibodycomprising a heavy chain polypeptide which is SEQ ID NO:1 and a lightchain polypeptide which is SEQ ID NO:2.

The invention also includes a kit for diagnosing HIT/HITT in a mammal.The kit comprises a) a positive control solution comprising a monoclonalantibody or a functional element thereof which binds specifically with aPF4/heparin complex, wherein the monoclonal antibody or the functionalelement thereof preferentially binds with the PF4/heparin complexrelative to the binding of the monoclonal antibody or the functionalelement thereof with either PF4 or heparin alone; and b) aninstructional material describing the use of the positive controlsolution for diagnosing HIT/HITT in a mammal.

In one aspect, the monoclonal antibody comprises a heavy chainpolypeptide having an amino acid sequence which shares at least about80% homology with SEQ ID NO:1 and a light chain polypeptide having anamino acid sequence which shares at least about 80% homology with SEQ IDNO:2.

In another aspect, the monoclonal antibody is a murine monoclonalantibody (KKO) comprising a heavy chain polypeptide of SEQ ID NO:1 and alight chain polypeptide of SEQ ID NO:2.

In a further aspect, the monoclonal antibody is a humanized antibody.

The invention also includes a kit for use in treating a mammal afflictedwith HIT/HITT. The kit comprises a) a composition comprising amonoclonal antibody or a functional element thereof which bindsspecifically with a PF4/heparin complex, wherein the monoclonal antibodyor the functional element thereof preferentially binds with thePF4/heparin complex relative to the binding of the monoclonal antibodyor the functional element thereof with either PF4 or heparin alone,wherein the monoclonal antibody or the functional element thereof ispresent in the composition in an amount effective to competitivelyinhibit the specific binding of a polyclonal antibody in the mammal tothe PF4/heparin complex; and b) an instructional material describing theuse of the composition for the treatment of HIT/HITT in the mammal.

In one aspect, the monoclonal antibody is a humanized antibody.

In another aspect, the monoclonal antibody comprises a heavy chainpolypeptide having an amino acid sequence which shares at least about80% homology with SEQ ID NO:1 and a light chain polypeptide having anamino acid sequence which shares at least about 80% homology with SEQ IDNO:2.

In a further aspect, the monoclonal antibody is a murine monoclonalantibody (KKO) comprising a heavy chain polypeptide of SEQ ID NO:1 and alight chain polypeptide of SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1, comprising FIGS. 1A-1C, is a series of graphs depicting thebinding of murine monoclonal antibody clones KKO and RTO to PF4. FIG. 1Adepicts the binding of KKO to microtiter wells coated with hPF4 (lineindicated by circles) or with a hPF4/heparin complex (line indicated bysquares). FIG. 1B depicts the binding of RTO to wells coated with hPF4(line indicated by squares) or with a hPF4/heparin complex (lineindicated with circles). FIG. 1C depicts the heparin dependence of KKOas measured by ELISA using wells coated with a fixed concentration ofhPF4 and varying concentrations of heparin. The data presented representthe mean±1 standard deviation of duplicate wells and are representativeof three independent measurements.

FIG. 2 is a graph depicting the reactivity of the murine monoclonalantibody KKO to hPF4/glycosaminoglycan complexes. The binding of KKOwith PF4 in a complex with glycosaminoglycans (GAGs) was assayed usingan ELISA method described herein. Sample wells were coated with a fixedconcentration of hPF4 (10 micrograms per milliliter) and the indicatedconcentration of either chondroitin sulfate A (ChSO₄A), chondroitinsulfate B (ChSO₄B), chondroitin sulfate C (ChSO₄C), dextran sulfate(DexSO₄) and heparan sulfate (HepSO₄). The data presented represent themean±1 standard deviation of duplicate wells and are representative oftwo independent measurements.

FIG. 3 is a graph depicting the binding of KKO at various concentrationsto human umbilical vein epithelial cells (HUVECs). The reactivity of KKOor an isotype control antibody (Con) to microtiter plates coated withHUVECs in the presence of either buffer or buffer containing hPF4(P),heparin (H), or hPF4/heparin complex (P/H). The data presented representthe mean of triplicate measurements and are representative of twoindependent measurements.

FIG. 4 is a graph depicting heparin dependent ¹⁴C-serotonin releasestimulated by KKO. ¹⁴C-labeled platelet rich plasma was incubated with80 micrograms per milliliter of either KKO or isotype control antibody(IC) in the presence of hPF4 (10 micrograms per milliliter) andsubsequently added to wells containing labeled platelets with theindicated concentrations of heparin. The data presented represent themean±1 standard deviation of triplicate measurements and arerepresentative of at least three independent measurements.

FIG. 5, comprising FIGS. 5A and 5B, is a pair of graphs which depict thecharacterization of the KKO binding site on the hPF4/heparin complex.FIG. 5A depicts the binding of KKO to single amino acid hPF4 mutantscomplexed to heparin as measured by ELISA. FIG. 5B depicts the resultsof competition studies of KKO binding to the hPF4/heparin complex usingHIT plasma at increasing concentrations with the results expressed aspercent of A₄₀₅ seen when no HIT plasma was included. The competitionstudies shown in black represent 4 different HIT patients “insensitive”to 3rd-domain mutations of hPF4, while the competition studies shown ingray represent HIT patients “sensitive” to 3rd-domain mutations of hPF4.The 50% level of reduction in A₄₀₅ is indicated as a dashed line.Results are the mean±1 standard deviation of three separate experimentsperformed in duplicate.

FIG. 6, comprising FIGS. 6A and 6B, is a pair of schematics depicting acomparison of the amino acid sequence of the murine monoclonalantibodies KKO and RTO. FIG. 6A depicts a comparison of the amino acidsequence of the heavy chain polypeptides of the antibodies. The upperpanel o FIG. 6A is KKO heavy chain (SEQ ID NO:11) and the lower panel isRTO heavy chain (SEQ ID NO:12). FIG. 6B depicts a comparison of theamino acid sequence of the light chain polypeptides. The upper panel ofFIG. 6B is KKO light chain (SEQ ID NO: 13) and the lower panel is RTOlight chain (SEQ ID NO: 14). Assigned variable region gene families andJ-gene segments are as indicated. Amino acid residue numbering andframework (FR) and complementarity-determining region (CDR) designationsare per Kabat et al., 1991 (“Sequences of Proteins of ImmunologicalInterest”, 5^(th) ed. Bethesda, National Institutes of Health). “>”indicates an amino acid residue encoded by a PCR primer.

FIG. 7, comprising FIGS. 7A-7D, is a listing of the amino acid andnucleotide sequences SEQ ID NOs: 1-4. FIG. 7A (SEQ ID NO:1) is the aminoacid sequence of the heavy chain polypeptide of the murine monoclonalantibody KKO. FIG. 7B (SEQ ID NO:2) is the amino acid sequence of thelight chain polypeptide of the murine monoclonal antibody KKO. FIG. 7C(SEQ ID NO:3) is the nucleotide sequence of an isolated nucleic acidwhich encodes the heavy chain polypeptide of KKO. FIG. 7D (SEQ ID NO:4)is the nucleotide sequence of an isolated nucleic acid which encodes thelight chain polypeptide of KKO.

FIG. 8 is a graph depicting the level of IL-8 secretion in the monocyticcell line U937 after incubation of the cells for 24 hours with either nostimulant (no antibody) or a stimulant selected from the groupconsisting of TNF-alpha (TNFa), the murine monoclonal antibody KKO(KKO), an isotype control antibody (TRA2b), sera from a patient with HIT(HIT sera) and control sera (control sera). The level of IL-8 secretionis expressed in picograms per milliliter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes compositions, kits and methods pertainingto a monoclonal antibody which binds specifically with a PF4/heparincomplex. The antibody is exemplified herein by a murine monoclonalantibody which exhibits several features which are critically similar tokey features of the polyclonal human antibodies which participate in thepathogenesis of heparin-induced thrombocytopenia and/or thrombosis(HIT/HITT). These features include preferential binding to a PF4/heparincomplex relative to binding of the antibodies with either PF4 or heparinalone, specific binding of the antibody to complexes of PF4 with othersulfated glycosaminoglycans (GAGs) besides heparin, and plateletactivation in the presence of the PF4/heparin complex. The exemplifiedmurine monoclonal antibody disclosed herein is the first knownmonoclonal antibody which preferentially binds the PF4/heparin complexrelative to either PF4 or heparin alone. Because this monoclonalantibody shares key serologic and functional properties with naturallyoccurring human polyclonal antibodies specific for the PF4/heparincomplex found in patients with HIT/HITT, the monoclonal antibody isuseful in methods for the diagnosis, treatment, and understanding of thepathogenesis of HIT/HITT in a mammal. Thus, the invention should not beconstrued as being limited solely to the antibody exemplified herein,but rather should be construed to include derivatives of this antibody,including humanized versions thereof as disclosed herein.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “pathogenesis of HIT/HITT” means one or more ofthe development of a symptom of HIT/HITT, the development of anunderlying physiological or molecular disorder which contributes toHIT/HITT, or the development of a pathological or deleteriousphysiological consequence of HIT/HITT in the mammal.

As used herein, the term “bind specifically with” or “specific binding”in the context of an antibody means to bind substantially to an antigen,without binding substantially to other molecules which are present withthe antigen.

As used herein, the term “preferentially binds” or to “bindpreferentially” means, in the context of an antibody, the binding of anantibody with a complex of more than one molecule with substantiallygreater affinity than the binding of the antibody with either of themolecules which comprise the complex alone.

As used herein, the phrase “to treat HIT/HITT” or “treating HIT/HITT” ina mammal means one or more of alleviating a symptom of, correcting anunderlying molecular or physiological disorder of, or reducing thefrequency or seventy of a pathological or deleterious physiologicalconsequence of HIT/HITT in the mammal. By way of example, and not bylimitation, the deleterious physiological consequences of HIT/HITTinclude stroke, myocardial infarction, arterial and venous thrombi,vascular gangrene and skin necrosis. Without wishing to be bound by anyone theory, it is suspected that a composition of the invention could,by way of example and not by limitation, effect treatment of HIT/HITT byone or more of blocking antigenic sites on a PF4/heparin complex whichare recognized by human polyclonal antibodies, binding Fc receptors andpreventing activation of platelets or endothelial cells, and creating ananti-idiotype response which could neutralize human polyclonalantibodies.

As used herein, the term “antibody” means an immunoglobulin moleculewhich is able to bind specifically to a specific epitope on an antigen.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources and can be immunoreactive portions (i.e.functional elements) of intact immunoglobulins. The antibodies in thecompositions and methods of the present invention may exist in a varietyof forms including, for example, polyclonal antibodies, monoclonalantibodies, Fv, Fab and F(ab)₂, as well as single chain antibodies andhumanized antibodies (Harlow et al., 1988, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

As used herein, the term “isolated polypeptide” refers to a polypeptidesegment or fragment which has been separated from sequences which flankit in a naturally occurring state, e.g., a polypeptide fragment whichhas been removed from the sequences which are normally adjacent to thefragment, e.g., the sequences adjacent to the fragment in a protein inwhich it naturally occurs. The term also applies to a polypeptide whichhas been substantially purified from other components which naturallyaccompany the polypeptide, e.g., proteins, RNA or DNA which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant polypeptide which is encoded by a nucleic acid incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g, as a cDNA or a genomic or cDNA fragment producedby PCR or restriction enzyme digestion) independent of other sequences.It also includes a recombinant polypeptide which is part of a hybridpolypeptide comprising additional amino acids. An isolated polypeptideof the invention is exemplified by the isolated polypeptides of SEQ IDNO:1 and SEQ ID NO:2 (the heavy and light chain polypeptides,respectively, of the murine monoclonal antibody KKO described herein).

As used herein, the term “isolated nucleic acid” refers to a nucleicacid segment or fragment which has been separated from sequences whichflank it in a naturally occurring state, e.g., a DNA fragment which hasbeen removed from the sequences which are normally adjacent to thefragment, e.g., the sequences adjacent to the fragment in a genome inwhich it naturally occurs. The term also applies to nucleic acids whichhave been substantially purified from other components which naturallyaccompany the nucleic acid, e.g., RNA or DNA or proteins, whichnaturally accompany it in the cell. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g, asa cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

As used herein, the term “recombinant polynucleotide” refers to apolynucleotide having sequences that are not naturally joined together.An amplified or assembled recombinant polynucleotide may be included ina suitable vector, and the vector can be used to transform a suitablehost cell. A recombinant polynucleotide may serve a non-coding function(e.g., promoter, origin of replication, ribosome-binding site, etc.) aswell. A host cell that comprises a recombinant polynucleotide isreferred to as a “recombinant host cell.” A gene which is expressed in arecombinant host cell wherein the gene comprises a recombinantpolynucleotide, expresses a “recombinant polypeptide.”

As used herein, the term “recombinant polypeptide” means a polypeptidewhich is produced upon expression of a recombinant polynucleotide.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

It will be appreciated, of course, that the peptides or polypeptides ofthe invention may incorporate amino acid residues which are modifiedwithout affecting activity. For example, the termini may be derivatizedto include blocking groups, i.e. chemical substituents suitable toprotect and/or stabilize the N- and C-termini from “undesirabledegradation”, a term meant to encompass any type of enzymatic, chemicalor biochemical breakdown of the peptide or polypeptide antibody orfragment thereof at its termini which is likely to affect the functionof the peptide or polypeptide in preferentially binding with aPF4/heparin complex relative to either PF4 or heparin alone, i.e.sequential degradation of the peptide or polypeptide at a terminal endthereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivity of the peptide or polypeptide antibody or fragment thereof. Forexample, suitable N-terminal blocking groups can be introduced byalkylation or acylation of the N-terminus. Examples of suitableN-terminal blocking groups include C₁-C₅ branched or unbranched alkylgroups, acyl groups such as formyl and acetyl groups, as well assubstituted forms thereof, such as the acetamidomethyl (Acm) group.Desamino analogs of amino acids are also useful N-terminal blockinggroups, and can either be coupled to the N-terminus of the peptide orused in place of the N-terminal reside. Suitable C-terminal blockinggroups, in which the carboxyl group of the C-terminus is eitherincorporated or not, include esters, ketones or amides. Ester orketone-forming alkyl groups, particularly lower alkyl groups such asmethyl, ethyl and propyl, and amide-forming amino groups such as primaryamines (—NH₂), and mono- and di-alkylamino groups such as methylamino,ethylamino, dimethylamino, diethylamino, methylethylamino and the likeare examples of C-terminal blocking groups. Descarboxylated amino acidanalogues such as agmatine are also useful C-terminal blocking groupsand can be either coupled to the peptide's C-terminal residue or used inplace of it. Further, it will be appreciated that the free amino andcarboxyl groups at the termini can be removed altogether from thepeptide to yield desamino and descarboxylated forms thereof withoutaffect on peptide activity in preferentially binding with a PF4/heparincomplex relative to either PF4 or heparin alone.

Other modifications can also be incorporated without adversely affectingthe biological activity of the peptide or polypeptide antibody orfragment thereof in preferentially binding with a PF4/heparin complexrelative to either PF4 or heparin alone. Such modifications include, butare not limited to, substitution of one or more of the amino acids inthe natural L-isomeric form with amino acids in the D-isomeric form.Thus, the peptide may include one or more D-amino acid residues, or maycomprise amino acids which are all in the D-form. Retro-inverso forms ofpeptides in accordance with the present invention are also contemplated,for example, inverted peptides in which all amino acids are substitutedwith D-amino acid forms.

Acid addition salts of the peptide or polypeptide antibody or fragmentthereof are also contemplated as functional equivalents. Thus, a peptidein accordance with the present invention treated with an inorganic acidsuch as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and thelike, or an organic acid such as an acetic, propionic, glycolic,pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric,citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic,p-toluenesulfonic, salicyclic and the like, to provide a water solublesalt of the peptide suitable for use in preferentially binding with aPF4/heparin complex relative to either PF4 or heparin alone.

The present invention also includes analogs of polypeptides or peptidesof the invention. Analogs can differ from naturally occurring proteinsor peptides by conservative amino acid sequence differences or by any ofthe modifications described herein or known in the art which do notaffect sequence, or by both. Modifications can be made as describedherein or by using any technique known to the skilled artisan.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups:

-   -   glycine, alanine;    -   valine, isoleucine, leucine;    -   aspartic acid, glutamic acid;    -   asparagine, glutamine;    -   serine, threonine;    -   lysine, arginine;    -   phenylalanine, tyrosine.        Modifications (which do not normally alter primary sequence)        include in vivo, or in vitro chemical derivatization of        polypeptides, e.g., acetylation, or carboxylation. Also included        are modifications of glycosylation, e.g., those made by        modifying the glycosylation patterns of a polypeptide during its        synthesis and processing or in further processing steps; e.g.,        by exposing the polypeptide to enzymes which affect        glycosylation, e.g., mammalian glycosylating or deglycosylating        enzymes. Also embraced are sequences which have phosphorylated        amino acid residues, e.g., phosphotyrosine, phosphoserine, or        phosphothreonine.

Also included in the invention are polypeptides which have been modifiedusing ordinary molecular biological techniques so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable for preferentially bindingwith a PF4/heparin complex relative to either PF4 or heparin alone.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

In any of the compositions and methods of the invention describedherein, one or more of a functional element, an analog, an epitope, anda chimeric peptide can be used in place of a polypeptide or peptideantibody or fragment thereof. This enables the use of a smaller peptidein the inventive compositions having similar or greater activity thanthe corresponding larger peptide or polypeptide of the invention.Methods for identifying a functional element, an epitope, an analog or achimeric polypeptide for any of the peptides or polypeptides of theinvention are described herein. By way of example and not by limitation,such functional elements, analogs, epitopes and chimeric peptides can beprepared using recombinant technology or isolated from natural sources.Alternatively, they can be prepared synthetically by using any peptidesynthesis method known in the art, such as a solid-phase peptidesynthesis method.

In one embodiment, the polypeptide of the invention is present in theinventive composition as an isolated polypeptide. The isolatedpolypeptide can be prepared by any method known to the skilled artisanfor preparing an isolated polypeptide.

For example, the isolated polypeptide of the invention can be obtainedby preparing and purifying a recombinant version of any of thepolypeptides of the invention described herein. Molecular biologytechniques for the preparation of recombinant polypeptides are wellknown in the art, and are described for example in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York; Ausubel et al., 1994, Current Protocols inMolecular Biology, John Wiley & Sons, New York), and Gerhardt et al.,eds., 1994, Methods for General and Molecular Bacteriology, AmericanSociety for Microbiology, Washington, D.C. Protein purification methodsare also well known in the art, and are described, for example inDeutscher et al. (ed., 1990, Guide to Protein Purification, HarcourtBrace Jovanovich, San Diego).

As used herein, an “analog” of a peptide or polypeptide antibody orfragment thereof means a peptide or polypeptide which has been modifiedfrom the naturally occurring peptide or polypeptide by any of themodifications described herein or known to the skilled artisan, butwhich still exhibits activity similar to the naturally occurring peptideor polypeptide in preferentially binding with a PF4/heparin complexrelative to either PF4 or heparin alone.

As used herein, a “chimeric peptide” or “chimeric polypeptide” means apeptide or polypeptide antibody or fragment thereof which comprises atleast a portion of a naturally occurring peptide or polypeptide and atleast a portion of a peptide or polypeptide with which it is normallynot found together with in nature, but which still exhibits activitysimilar to the naturally occurring peptide or polypeptide of theinvention in preferentially binding with a PF4/heparin complex relativeto either PF4 or heparin alone.

As used herein, a “functional element” of a peptide or a polypeptideantibody or fragment thereof means a portion of a peptide or polypeptideof the invention which participates in the pathogenesis of HIT/HITT. Byway of example and not by limitation, the functional element can be apeptide, a peptide analog or an epitope of a polypeptide of theinvention, a chimeric peptide thereof or an analog of a chimeric peptidethereof. A method of identifying a functional element of a peptide orpolypeptide of the invention is described herein.

As used herein, an “epitope” of a peptide or a polypeptide of theinvention means a portion of a peptide or polypeptide of the inventionwhich is exposed at the surface of the peptide or polypeptide of theinvention or which is accessible to an antigen (i.e. PF4/heparin complexor PF4/glycosaminoglycan complex) of a peptide or polypeptide of theinvention.

As used herein, the term “vector” means a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into cells, such as, for example, polylysine compounds,liposomes, and the like. Examples of viral vectors include, but are notlimited to, adenoviral vectors, adeno-associated viral vectors,retroviral vectors, and the like.

As used herein, the term “expression vector” refers to a vectorcomprising a recombinant polynucleotide comprising expression controlsequences operatively linked to a nucleotide sequence to be expressed.An expression vector comprises sufficient cis-acting elements forexpression; other elements for expression can be supplied by the hostcell or in an in vitro expression system. Expression vectors include allthose known in the art, such as cosmids, plasmids (e.g., naked orcontained in liposomes) and viruses that incorporate the recombinantpolynucleotide.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

Description

The invention relates to compositions, kits and methods comprising amonoclonal antibody which shares key functional properties with thepolyclonal antibodies which participate in the pathogenesis of heparininduced thrombocytopenia/thrombosis (HIT/HITT) in a mammal. Themonoclonal antibody is exemplified herein by a murine monoclonalantibody which preferentially binds with a PF4/heparin complex relativeto the binding of the antibody with either PF4 or heparin alone. Themonoclonal antibody also binds specifically with PF4 in a complex withother glycosaminoglycans besides heparin, and also activates platelets.The monoclonal antibody is thus useful in methods for diagnosing andtreating HIT/HITT in a mammal. A humanized version of the monoclonalantibody is also included, along with a process for humanizing themonoclonal antibody.

The invention includes a composition comprising a monoclonal antibodywhich is capable of binding specifically with a PF4/heparin complex. Themonoclonal antibody of the invention preferentially binds with thePF4/heparin complex relative to the binding of the antibody with eitherPF4 or heparin alone. The monoclonal antibody can be any type ofmonoclonal antibody known to the skilled artisan or yet to be known. Byway of example and not by limitation, such monoclonal antibody typesinclude humanized antibodies, synthetic antibodies and phage displayedantibodies.

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev, in Immunol. 12(3,4):125-168) and thereferences cited therein. Further, the antibody of the invention may be“humanized” using the technology described in Wright et al., (supra) andin the references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77(4):755-759).

In one embodiment, the monoclonal antibody of the invention is ahumanized monoclonal antibody. Humanized monoclonal antibodies areuseful for reducing the immunogenicity of a monoclonal antibody in ahuman.

Several approaches are known in the art to reduce the immunogenicity ofmurine monoclonal antibodies in humans. One approach is to constructchimeric molecules which have murine variable regions (Fab), whichretain antigenic specificity, connected to human constant regions (Fc).This can be accomplished using established techniques in geneticengineering including the following: 1) transfection of human and murinechain gene constructs into a non-secreting myeloma cell line (Morrisonet al., 1984, Proc. Natl. Acad. Sci. USA, 81: 6851-6855); or 2) genetargeting through homologous recombination (Fell et al., 1989, Proc.Natl. Acad. Sci. USA. 86: 8507-8511). However, immune responses to thevariable region of the chimeric antibodies still occur, and thus limittheir therapeutic use in some cases (Knight et al., 1995, Mol. Immunol.32, 1271-1281).

In order to further reduce the human immune response to murinemonoclonal or chimeric antibodies, techniques for humanizing antibodieshave been developed (Jones et al., 1986, Nature 321: 522-525; Reichmanet al., 1988, Nature 332: 323-327). These techniques involve geneticgrafting of the complementary-determining regions (CDR) of the murineantibody onto the human heavy and light chain framework residues. Theoriginal technique described by Reichman et. al. involves producing ahuman variable region with the desired specificity of the murinehypervariable region. Murine hypervariable (HV) primers with sequencescomplementary to the murine HV region are synthesized with flankinghuman framework sequences corresponding to the human HV region. Thesemurine HV primers, in addition to nucleotides and DNA polymerase, areadded to a plasmid containing the human variable region gene. Thisreaction results in a humanized murine variable gene (huV), containinghuman framework regions and murine HV regions. The humanized variableregion is next inserted into a plasmid containing a human constantregion gene.

This procedure was employed to create humanized heavy and light chainswhich were then cotransfected into a non-secreting myeloma cell line.Subsequent refinements of this basic procedure have resulted in aprocess for producing humanized antibodies with affinities andbiological properties comparable to that of the parent murine monoclonalantibody (Emery et al., 1995, Strategies for humanizing antibodies, In:Antibody Engineering, 2^(nd) ed., Ed. Borrebaeck CAK) pp 159-183, OxfordUniversity Press, Oxford). This technology can be similary utilized tocreate functional humanized Fab′ fragments in bacterial cells (Carteret. al., 1992. Bio/Technology, 10: 163-168).

For the purposes of the present invention, the latter technique ofhumanizing antibodies using CDR grafting to generate humanizedmonoclonal antibodies or Fab derivatives thereof is the preferredtechnique for reducing the immunogenicity of murine monoclonalantibodies in humans.

Antibodies displayed at the surface of a bacteriophage are alsocontemplated by the antibodies of the invention. Bacteriophage whichencode the desired antibody may be engineered such that the protein isdisplayed on the surface thereof in such a manner that it is availablefor binding to its corresponding binding protein, e.g., the antigenagainst which the antibody is directed. Thus, when bacteriophage whichexpress a specific antibody are incubated in the presence of a cellwhich expresses the corresponding antigen, the bacteriophage will bindto the cell. Bacteriophage which do not express the antibody will notbind to the cell. Such panning techniques are well known in the art andare described for example, in Wright et al., (supra).

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired protein to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al., 1991, J. Mol. Biol. 222:581-597. Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.1995, J. Mol. Biol. 248:97-105).

In one embodiment, the monoclonal antibody of the invention comprises aheavy chain polypeptide having an amino acid sequence which shares atleast about 80% homology with SEQ ID NO:1 (FIG. 7A). Preferably, theheavy chain polypeptide is about 85% homologous, more preferably about90% homologous, even more preferably about 95% homologous, and mostpreferably about 99% homologous to the heavy chain polypeptide of SEQ IDNO:1. Even more preferably, the monoclonal antibody of the inventioncomprises a heavy chain polypeptide which is SEQ ID NO:1. Also, in thisembodiment the monoclonal antibody of the invention comprises a lightchain polypeptide having an amino acid sequence which shares at leastabout 80% homology with SEQ ID NO:2 (FIG. 7B). Preferably, the lightchain polypeptide is about 85% homologous, more preferably about 90%homologous, even more preferably about 95% homologous, and mostpreferably about 99% homologous to the light chain polypeptide of SEQ IDNO:2. Even more preferably, the monoclonal antibody of the inventioncomprises a light chain polypeptide which is SEQ ID NO:2.

The determination of percent homology (i.e. percent identity) describedherein between two amino acid or nucleotide sequences can beaccomplished using a mathematical algorithm. For example, a mathematicalalgorithm useful for comparing two sequences is the algorithm of Karlinand Altschul (1990, Prc. Natl. Acad. Sci. USA 87:2264-2268), modified asin Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877).This algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed,for example, at the National Center for Biotechnology Information (NCBI)world wide web site having the universal resource locator“www.ncbi.nlm.nih.gov/BLAST”. Blast nucleotide searches can be performedwith NBLAST program (designated “blastn” at NCBI web site), using thefollowing parameters: gap penalty=5; gap extension penalty=2; mismatchpenalty=3; match reward=1; expectation value 10.0; and word size=11 toobtain nucleotide sequences homologous to a nucleic acid describedherein. BLAST protein searches can be performed with XLBLAST Program(designated “blastn” at the NCBI web site) or the NCBI “blastp” program,using the following parameters: expectation value 10.0, BLOSUM62 scoringmatrix to obtain amino acid sequences homologous to a protein moleculedescribed herein

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an integrated search which detects distant relationships betweenmolecules (id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped Blast, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See the world wide web having theuniversal locator www.ncbi.nlm.nih.gov.

The percent identity between two amino acid or nucleotide sequences canbe determined using techniques similar to those described above, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

In a preferred aspect, the monoclonal antibody of the invention is amurine monoclonal antibody comprising the heavy chain polypeptide of SEQID NO: 1 and the light chain polypeptide of SEQ ID NO:2, and is referredto herein as “KKO”.

Deposit

Hybridoma KKO, was deposited on Jul. 30, 2004, with the American TypeCulture Collection (ATCC) of 10801 University Blvd., Manassas, Va.,20110-2209 USA, and accorded ATCC accession number PTA-6133.

In one embodiment, the monoclonal antibody of the invention is capableof activating platelets in the presence of a PF4/heparin complex.

In another embodiment, the monoclonal antibody of the invention is alsocapable of binding specifically with a complex of PF4 and aglycosaminoglycan (GAG) which is not heparin. Preferably, the monoclonalantibody of the invention preferentially binds with the complex ofPF4/GAG relative to the binding of the antibody with either PF4 or theGAG alone. Non-limiting examples of such other glycosaminoglycansinclude chondroitin sulfates A, B and C, heparan sulfate, dextransulfate and low molecular weight heparin. Other glucanated sulfatesknown in the art as recognized by human polyclonal HIT/HITT antibodiesare included within the GAGs of the invention, and are discussed, forexample, in Greinacher et al., 1995, Thrombosis and Haemostasis,74:886-892.

The invention also includes a composition comprising an isolated nucleicacid, wherein the isolated nucleic acid encodes one or more of a heavychain and a light chain polypeptide of a monoclonal antibody which iscapable of binding specifically with a PF4/heparin complex. The antibodycomprised of the heavy and light chain polypeptides encoded by the oneor more isolated nucleic acids of the invention preferentially bindswith the PF4/heparin complex relative to the binding of the antibodywith either PF4 or heparin alone.

The isolated nucleic acid which encodes the heavy chain polypeptide hasa nucleotide sequence which shares at least about 80% homology with SEQID NO:3 (FIG. 7C). Preferably, the isolated nucleic acid which encodesthe heavy chain polypeptide is about 85% homologous, more preferablyabout 90% homologous, even more preferably about 95% homologous, andmost preferably about 99% homologous to the isolated nucleic acidcorresponding to SEQ ID NO:3. Even more preferably, the isolated nucleicacid which encodes the heavy chain polypeptide is the isolated nucleicacid of SEQ ID NO:3.

The isolated nucleic acid which encodes the light chain polypeptide hasa nucleotide sequence which shares at least about 80% homology with SEQID NO:4 (FIG. 7D). Preferably, the isolated nucleic acid which encodesthe light chain polypeptide is about 85% homologous, more preferablyabout 90% homologous, even more preferably about 95% homologous, andmost preferably about 99% homologous to the isolated nucleic acidcorresponding to SEQ ID NO:4. Even more preferably, the isolated nucleicacid which encodes the light chain polypeptide is the isolated nucleicacid of SEQ ID NO:4.

In embodiments of the invention where the inventive compositioncomprises an isolated nucleic acid, the isolated nucleic acid ispreferably present in an amount effective to transform a mammalian cellto provide expression of the monoclonal antibody at a level ofexpression effective to result in production of the monoclonal antibodyin the mammal at a level sufficient to provide competitive inhibition ofthe specific binding of a polyclonal antibody within the mammal with aPF4/heparin complex.

The isolated nucleic acid can be either alone as a “naked” nucleic acid,such as a linearized nucleic acid, or as a component of any type ofvector suitable for transfecting a mammalian cell described herein orknown in the art. Preferably, the isolated nucleic acid is a recombinantpolynucleotide component of a viral or plasmid expression vectorsuitable for transfecting a mammalian cell, and is operably linked tothe appropriate regulatory elements to provide a high level ofexpression of the transgene once a targeted mammalian cell istransformed with the isolated nucleic acid. Examples of preferredvectors include adenovirus, retrovirus, lentivirus and adeno-associatedvirus vectors. Techniques for using such vectors to transfect amammalian cell are known in the art. Appropriate promoter/regulatoryelements to be included in the vector used will be apparent to theskilled artisan.

When any one of the polypeptides of the invention are to be administeredto a mammal or to a tissue of a mammal in a method of the invention forthe purpose of exerting a beneficial effect in the mammal, the inventionshould be construed to include delivery of the polypeptide via deliveryof an isolated nucleotide sequence encoding the peptide. Expression ofthe peptide from the nucleotide sequence so delivered to the desiredtissue is effective administration of the peptide to the cell or tissue.

In another embodiment, the inventive composition is in the form of apharmaceutical composition comprising a pharmaceutically acceptablecarrier. Such a pharmaceutical composition may consist of the inventivecomposition alone as the active ingredient, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the inventive composition as the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of apharmaceutically acceptable salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “pharmaceutically acceptable salt” means a saltform of the active ingredient which is compatible with any otheringredients of the pharmaceutical composition, which is not deleteriousto the subject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to any mammal. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various mammals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor any route of administration known to the skilled artisan, including,by way of example and not by limitation, oral, parenteral, topical,ocular, inhalation, intrauterine, intravesicular, intraurethral andbuccal routes of administration. The pharmaceutical composition can beadministered to a mammal by any route of administration known to theskilled artisan, such as those described above, and by any method ofadministering a pharmaceutical composition to a mammal known in the art.A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Controlled or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the composition of the invention which may beadministered to an animal, preferably a human, range in amount from 1microgram to about 100 grams per kilogram of body weight of the animal.While the precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal and the routeof administration. Preferably, the dosage of the composition will varyfrom about 1 mg to about 10 g per kilogram of body weight of the animal.More preferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The composition may be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenless frequently, such as once every several months or even once a yearor less. The frequency of the dose will be readily apparent to theskilled artisan and will depend upon any number of factors, such as, butnot limited to, the type and severity of the disease being treated, thetype and age of the animal, etc.

Any of the pharmaceutical compositions of the invention may furthercomprise one or more compounds known in the art to enhance andfacilitate drug administration.

Other possible formulations, such as nano particles, liposomes, resealederythrocytes, and immunologically based systems such as antibodytargeting systems may also be used to administer the inventivecomposition according to the methods of the invention described herein.

In one embodiment, the composition of the invention comprising themonoclonal antibody is in the form of a pharmaceutical composition.

The invention also includes a method of making a humanized monoclonalantibody which is capable of binding specifically with a PF4/heparincomplex. The monoclonal antibody preferentially binds with thePF4/heparin complex relative to the binding of the antibody with eitherPF4 or heparin alone.

The method comprises obtaining a monoclonal antibody which is capable ofbinding specifically with a PF4/heparin complex. The monoclonal antibodypreferentially binds with the PF4/heparin complex relative to thebinding of the antibody with either PF4 or heparin alone. The monoclonalantibody can be obtained from any source, either natural or synthetic.When the monclonal antibody is prepared synthetically, any method knownto the skilled artisan for the preparation of a monoclonal antibody maybe used. An example of such a method is described herein in theExperimental Examples.

The humanized monoclonal antibody preferentially binds the PF4/heparincomplex relative to the binding of the antibody with either PF4 orheparin alone.

In one aspect, the monoclonal antibody used in the method comprises aheavy chain polypeptide having an amino acid sequence which shares atleast about 80% homology with the polypeptide of SEQ ID NO:1, andcomprises a light chain polypeptide having an amino acid sequence whichshares at least about 80% homology with the polypeptide of SEQ ID NO:2.Preferably, the monoclonal antibody used in the method comprises a heavychain polypeptide having the amino acid sequence of SEQ ID NO:1, andcomprises a light chain polypeptide having the amino acid sequence ofSEQ ID NO:2.

The invention also includes a method of diagnosing heparin-inducedthrombocytopenia/thrombosis (HIT/HITT) in a mammal. The method comprisesassessing the level of a polyclonal antibody which binds specificallywith a PF4/heparin complex in a sample obtained from the mammal andcomparing the level assessed with a positive control sample forHIT/HITT. The positive control sample comprises any of the monoclonalantibodies or fragments thereof described in the inventive compositions.When the level or concentration of the polyclonal antibody in the sampleobtained from the mammal is statistically similar to the level of themonoclonal antibody present in the positive control sample, thenHIT/HITT is diagnosed in the mammal. The mammal can be any mammal but ispreferably a human.

The method comprises obtaining a sample of a bodily fluid or tissue fromthe mammal. By way of example, and not by limitation, the bodily fluidor tissue can be a blood sample, a serum sample, a biopsy sample, acerebrospinal fluid sample, a urine sample, an ascites sample, a pleuraleffusion sample, a clot tissue sample, or any other tissue sample.

The method also includes assessing the level of a polyclonal antibody inthe sample which binds specifically with a PF4/heparin complex. Thepolyclonal antibody preferentially binds with the PF4/heparin complexrelative to the binding of the polyclonal antibody with either PF4 orheparin alone. The level of the polyclonal antibody can be assessed byany method known to the skilled artisan for assessing the level orconcentration of an antibody in a sample.

Preferred methods for assessing the level of the polyclonal antibody inthe sample include an ELISA assay, a Western Blotting assay, a serotoninrelease assay, a platelet aggregation assay, a lumi-aggregometry assay,and a flow cytometry assay. Such methods are known in the art ordescribed herein. Briefly, an ELISA assay can be used in which theantigen (PF4/heparin complex) is coated on microtiter plates and thepresence of antigen-specific antibodies is detected usingenzyme-labelled anti-human or anti-mouse antibodies (See, Arepally etal., 1995. Am. J. Clin. Pathol., 104:648). A Western blotting assay canbe used in which the PF4/heparin complex is the antigen (See, Harlow etal., 1988, Antibodies: A Laboratory Manual, N.Y., Cold Spring HarborLaboratory, 479-504). A serotonin release assay can be used in whichplatelets are loaded with radiolabeled serotonin and incubated in thepresence of heparin or buffer and HIT/HITT antibodies. A positive resultis indicated by platelet activation and the release of radiolabeledserotonin into the supernatant in the presence of heparin, but not inthe presence of buffer (See, Sheridan et al., 1986, Blood, 67:27-30).

Variations upon the platelet activation assay include the plateletaggregation assay, which employs a platelet aggregometer to opticallydetect the activation of platelets, the heparin induced plateletactivation assay, which employs a visual endpoint of transparency fordetecting platelet activation, and lumi-aggregometry, which measuresluminescence detected via ATP release as an endpoint for plateletactivation (Stewart et al., 1995, British J. Haematol. 91:173-177).

Additionally, flow cytometry assays can be used to assess the level ofthe polyclonal antibody in the sample. Briefly, fluorescent labeledantibodies to platelets are used to measure platelet activation byHIT/HITT antibodies in the presence of heparin or buffer using a flowcytometer instrument. The generation of activated platelets is detectedby the binding of activation-specific markers (Tomer, A., 1997, Br. J.Haematol., 98:648-656) or by microparticle generation (Lee et al., 1996,Br. J. Haematol., 95:724-731).

Other methods known in the art for assessing the level or concentrationof a polyclonal antibody in a sample can be used, such as, by way ofexample and not by limitation, chromatographic methods or otherimmunological methods (See, for example, Harlow et al., 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor, New York). Forexample, particle-gel immunoassay methods can be used which employ aparticle gel immunoassay commonly employed in transfusion medicine. Redhigh density polystyrene beads coated with human PF4/heparin complexesare exposed to HIT/HITT antibodies in a reaction chamber containing abuffered sephacryl gel matrix. The beads are centrifuged andagglutination at the top of the dispersed gel is interpreted as positivedetection. Enzyme immunoassay (EIA) methods can also be used. An EIA isa fluid phase assay which measures the binding of HIT/HITT antibodies tobiotinylated-PF4/heparin complexes in solution (See, e.g. Newman et al.,1998, Thrombosis and Haemostasis. 80:292-297).

The method also includes comparing the level of the polyclonal antibodyin the sample assessed as described above with the level of a monoclonalantibody which is specific for the PF4/heparin complex in a positivecontrol sample for HIT/HITT. The level of the monoclonal antibodypresent in the positive control sample can be assessed by any methodknown to the skilled artisan for measuring the level or concentration ofan antibody in a sample. Preferred methods for assessing the level ofthe monoclonal antibody in the sample include an ELISA assay, aserotonin release assay, a platelet aggregation assay, alumi-aggregometry assay, and a flow cytometry assay.

Other methods known in the art for assessing the level or concentrationof a monoclonal antibody such as, by way of example and not bylimitation, chromatographic methods or other immunological methods canalso be used to detect the level of the monoclonal antibody in thepositive control sample.

The method also includes determining from the level of the polyclonalantibody and the level of the monoclonal antibody assessed as describedabove, whether the level of the polyclonal antibody in the sampleobtained from the mammal is statistically similar to the level of themonoclonal antibody in the positive control sample for HIT/HITT. Thedetermination of whether the levels of the monoclonal and polyclonalantibodies are statistically similar can be made by any statisticalmethod known to the skilled artisan. The ordinarily skilled artisan willbe aware that the percent variation from the positive control whichcould be used as a cut-off point for the diagnosis of HIT/HITT dependsupon the type of method used to assess the levels of antibodies in thesample, and is calibrated using the positive control antibodies andnegative controls (i.e. from normal donors or blank controls). Eachassay method has separate criteria for determining a positive result,and tropically employs three standard deviations from negative controlsfor the positive control to be considered positive. Each of the methodsdiscussed above for assessing antibody levels in a sample discuss in thecited references the criteria for a positive reaction for the particulartype of test.

The range of concentrations appropriate for use as the concentration ofthe monoclonal antibody in a positive control sample for HIT/HITT willvary from about 1 microgram per milliliter to about 1 milligram permilliliter. Preferably, the concentration of the monoclonal antibodyranges from about 50 to about 200 micrograms per milliliter, and morepreferably, from about 80 to about 150 micrograms per milliliter.

When the level or concentration of the polyclonal antibody present inthe original sample of a body fluid or tissue obtained from the mammalis statistically similar to the level of the monoclonal antibody presentin the positive control sample, then HIT/HITT is diagnosed in themammal.

In another embodiment, the method does not include diagnosing HIT/HITTin a mammal, but instead involves simply assessing the level of thepolyclonal antibody in the sample obtained from the mammal. In thisembodiment, the method comprises obtaining a sample of a bodily fluid ortissue from the mammal. The bodily fluid or tissue can be any of thosedescribed herein.

The method also includes assessing the level in the sample of apolyclonal antibody which binds specifically with a PF4/heparin complexand which preferentially binds with the PF4/heparin complex relative tothe binding of the polyclonal antibody with either PF4 or heparin alone.The level of the polyclonal antibody in the sample can be assessed byany method known to the skilled artisan or described herein forassessing the level or concentration of an antibody in a sample.

The method also includes comparing the level of the polyclonal antibodyin the sample assessed as described above with the level of a monoclonalantibody in a reference standard containing the monoclonal antibody. Themonoclonal antibody is specific for the PF4/heparin complex andpreferentially binds with the PF4/heparin complex relative to thebinding of the monoclonal antibody with either PF4 or heparin alone. Thelevel of the monoclonal antibody in the reference standard can beassessed by any method known to the skilled artisan or described hereinfor measuring the level or concentration of an antibody in a sample.Preferred methods for assessing the level of the monoclonal antibody inthe reference standard include an ELISA assay, a serotonin releaseassay, a platelet aggregation assay, a lumi-aggregometry assay, and aflow cytometry assay.

The monoclonal antibody can be any of the monoclonal antibodiesdescribed herein in the inventive compositions, or a functional elementor fragment thereof. A preferred monoclonal antibody is the murinemonoclonal antibody KKO, or a functional element, fragment or humanizedversion thereof.

The method also includes determining, from the comparison describedabove, the level of the polyclonal antibody in the sample obtained fromthe mammal, thereby determining the level of a polyclonal antibody inthe mammal which binds specifically with a PF4/heparin complex and whichpreferentially binds with the PF4/heparin complex relative to thebinding of the polyclonal antibody with either PF4 or heparin alone.

The invention also includes a method of identifying a functional elementof an antibody, wherein the functional element participates in thepathogenesis of HIT/HITT in a mammal. The method comprises preparing oneor more deletion mutants of a monoclonal antibody which bindsspecifically with a PF4/heparin complex, and which preferentially bindswith the PF4/heparin complex relative to the binding of either PF4 orheparin alone. The deletion mutant can be a polypeptide having either adeletion or a substitution mutation. Methods of preparing deletion orsubstitution mutants of polypeptides and antibodies are known in the art(See, for example, Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York and Ausubelet al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons,New York). The one or more deletion mutants prepared lack a portion ofthe amino acid sequence of the Fab region of the monoclonal antibody.

The method also includes assessing the ability of each of the deletionmutants so prepared to bind specifically with a PF4/heparin complex, andto preferentially bind with the PF4/heparin complex relative to thebinding of the deletion mutant with either PF4 or heparin alone. Theassessment of such binding can be performed using any immunological orother assay know to the skilled artisan or described herein forassessing the binding of an antibody to an antigen. Examples of suchmethods are described herein or known in the art. For example, thedetermination of loss or gain of the ability of a deletion mutant tobind specifically with a PF4/heparin complex can be performed bymeasuring the association and disassociation constants forantigen-antibody interactions (See, e.g., Tomlinson, G. 1988, Trends inPharm. Sci., 9:159-162).

The method also includes identifying from the binding ability assessedas described above for each of the deletion mutants, one or more of thedeletion mutants which does not preferentially bind with the PF4/heparincomplex relative to the binding of the deletion mutant with either PF4or heparin alone. Based upon the one or more deletion mutants soidentified, the corresponding deleted portion of the amino acid sequenceof the monoclonal antibody which participates in the preferentialbinding of the antibody with the PF4/heparin complex is determined. Bydetermining which portion of the amino acid sequence of the monoclonalantibody participates in preferential binding with a PF4/heparincomplex, a functional element of an antibody which participates in thepathogenesis of HIT/HITT in a mammal is identified.

In addition to the method discussed above, other methods known in theart for identifying a functional element of an antibody can be used. Forexample, anti-peptide antibodies, deletion/substitution mutants,chimeric antibodies and other peptide inhibition methods can be used toidentify a functional element of the monoclonal antibody of theinvention which participates in the pathogenesis of HIT/HITT in amammal. A functional element of the antibody can be identified throughthe construction of chimeric or humanized antibodies which are derivedfrom two species, such as human and mouse. Methods for constructing andevaluating chimeric antibodies are discussed above.

The invention also includes a method of treating HIT/HITT in a mammal,preferably, a human. The method comprises administering to the mammal acomposition comprising the monoclonal antibody of the invention, or afunctional element thereof, which binds specifically with a PF4/heparincomplex, and which preferentially binds with the PF4/heparin complexrelative to the binding of the monoclonal antibody with either PF4 orheparin alone. The composition is administered in an amount effective tocompetitively inhibit the specific binding of a polyclonal antibody inthe mammal to a PF4/heparin complex, thus inhibiting the specificbinding of the polyclonal antibody in the mammal with the PF4/heparincomplex, thereby treating HIT/HITT in the mammal.

In the method of the invention, any of the inventive compositionsdisclosed herein can be administered to the mammal to be treated by anyroute of administration known in the art or described herein.Preferably, the inventive composition is administered parenterally (i.e.intravenously or intramuscularly).

In one aspect, the inventive composition is administered to the mammalin the form of a pharmaceutical composition. The pharmaceuticalcomposition can be any of the pharmaceutical compositions describedherein.

In the method of the invention, the use of a derivatized fragment of amonoclonal antibody (i.e. such as a derivatized Fab or F(ab)₂ fragmentinstead of the entire antibody molecule) in the inventive composition ispreferred, since such fragments will retain antigen specificity withoutcausing effector cell activation, such as platelet activation throughthe Fc portion of the antibody. Fab and F(ab)₂ fragments can be preparedand derivatized using immunological methods well known in the art (See,for example, Harlow et al., 1988, Antibodies: A Laboratory Manual. NY,Cold Spring Harbor, 479-504). Other therapeutically beneficialmodifications include altering the IgG subclass isotype of a murine orhumanized antibody of the invention using recombinant technology inorder to abolish effector cell activation.

In the method of the invention, competitively inhibiting the specificbinding of the polyclonal antibody means to substantially reduce, impedeor inhibit the specific binding of the polyclonal antibody in the mammalto a PF4/heparin complex by the specific binding of a monoclonalantibody of the invention or a fragment or functional element thereof tothe PF4/heparin complex. The ordinarily skilled artisan will be aware ofthe minimum percent inhibition that must be exhibited by theadministered monoclonal antibody of the invention to be consideredsubstantial competitive inhibition of the polyclonal antibody which isefficacious in the treatment of HIT/HITT in a mammal.

The inventive composition can be provided to the mammal either alone in“naked” form, for example as an isolated polypeptide or an isolatednucleic acid encoding the same, or it can be formulated in a vehiclesuitable for delivery, such as, by way of example and not by limitation,in a complex with a cationic molecule or a liposome forming lipid, in avector, or as a component of a pharmaceutical composition. Such vehiclesare well known to the skilled artisan. The composition is preferably ahumanized antibody, or a nucleic acid encoding the sane.

In one embodiment, the monoclonal antibody in the compositionadministered to the mammal is a monoclonal antibody comprising a heavychain polypeptide having an amino acid sequence which shares at leastabout 80% homology with SEQ ID NO:1 and a light chain polypeptide havingan amino acid sequence which shares at least about 80% homology with SEQID NO:2. Preferably, the monoclonal antibody is a humanized monoclonalantibody.

The invention also includes a method of identifying a compound which isa modulator of the specific binding of an antibody to a PF4/heparincomplex. Such a compound is useful for treating HIT/HITT in a mammal, byway of example and not by limitation, by decreasing the level ofspecific binding of a polyclonal antibody in the human to a PF4/heparincomplex, wherein the specific binding of the polyclonal antibody to thePF4/heparin complex is a factor in the pathogenesis of HIT/HITT, or byincreasing the level of specific binding of a monoclonal antibody of theinvention or a functional element or fragment thereof to the PF4/heparincomplex in a mammal, thereby competitively inhibiting the binding of apolyclonal antibody in the mammal to the PF4/heparin complex, which is afactor in the pathogenesis of HIT/HITT. The compound can be any type ofcompound, and includes by way of example and not by limitation, apeptide, a polypeptide, an oligonucleotide, a nucleic acid, a drug, aligand and a receptor. Modulating the specific binding of the antibodyto the PF4/heparin complex can mean either to increase or to decreasethe level of specific binding or binding affinity of the antibody to thePF4/heparin complex. The compound can operate by either directly orindirectly increasing or decreasing the level of specific binding orbinding affinity of the antibody to the PF4/heparin complex by any modeof action. By way of example and not by limitation, the compound can actdirectly by competing for binding sites upon one or more of theantibody, PF4 or heparin, or can act indirectly by acting through asignal transduction pathway or another molecule which serves as theprimary effector or through any other biological process.

The method comprises contacting the compound with the antibody and thePF4/heparin complex. The compound, the antibody and the PF4/heparincomplex can be contacted in any chronological order, and need not bepresent together simultaneously. The components can be contacted in aliquid medium such as a solution, or on a solid surface, such as anmicrowell plate or a bead. A sufficient period of time is permitted forspecific binding between the antibody and the PF4/heparin complex.

The method also comprises assessing the effect of the compound upon thelevel of specific binding of the antibody to the PF4/heparin complex.The level of specific binding can be assessed by any method known in theart or described herein for assessing the specific binding or bindingaffinity of an antibody to an antigen. In order to determine the effectof the compound, a comparison is made between the specific binding orbinding affinity of the antibody to the PF4/heparin complex in thepresence and the absence of the compound.

The effect of the compound assessed as described above is an indicationof whether the compound is a modulator of the specific binding of theantibody to the PF4/heparin complex. If the compound eithersubstantially increases or substantially decreases the level of specificbinding of the antibody to the PF4/heparin complex, then a compound isidentified which is a modulator of the specific binding of the antibodyto the PF4/heparin complex. It will be apparent to the skilled artisanhow to determine whether such increases or decreases in the level ofspecific binding are substantial.

In one embodiment, the antibody is a monoclonal antibody. The monoclonalantibody can be any monoclonal antibody or functional element, fragment,or humanized version thereof described herein in the inventivecompositions. A preferred monoclonal antibody is the murine monoclonalantibody KKO or a functional element, fragment, or humanized versionthereof.

In another embodiment, the antibody is a polyclonal antibody. Thepolyclonal antibody can be any polyclonal antibody which is capable ofspecific binding to a PF4/heparin complex and preferentially binding thePF4 heparin complex relative to either PF4 or heparin alone.

The invention also includes a kit for diagnosing HIT/HITT in a mammal.The kit comprises a positive control solution comprising a monoclonalantibody of the invention, or a functional element thereof, which bindsspecifically with a PF4/heparin complex. The monoclonal antibody can beany of the monclonal antibodies described herein in the inventivecompositions. Preferably, the monoclonal antibody is a humanizedmonoclonal antibody.

The positive control solution comprises the monoclonal antibody of theinvention or a functional element thereof in an amount ranging fromabout 1 picogram per milliliter to about 10 milligrams per milliliter,preferably from about 1 microgram per milliliter to about 200 microgramsper milliliter. The skilled artisan will be aware of appropriatevariations in concentration for the monoclonal antibody in the positivecontrol sample depending upon the sensitivity of the assay method usedto assess levels of the antibody. For example, in the ELISA assaydescribed herein, a preferred concentration range for the monoclonalantibody in the positive control ranges from about 0.1 to about 40micrograms per milliliter, and in the serotonin release assay describedherein a preferred concentration range for the monoclonal antibody inthe positive control ranges from about 75 to about 150 micrograms permilliliter.

In addition to the monoclonal antibody of the invention, the positivecontrol solution may further comprise one or more of a suitable bufferfor suspending a monoclonal antibody, such as a phosphate-bufferedsaline buffer or a tris buffer, a stabilizer such as a glycerol solutionor bovine serum albumin, and a preservative such as sodium azide.

The kit also includes an instructional material describing the using ofthe positive control solution for diagnosing HIT/HITT in a mammal usingthe inventive method described herein for diagnosing HIT/HITT in amammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expressing which can beused to communicate the usefulness of the composition of the inventionin the kit for diagnosing HIT/HITT in a mammal. Optionally, oralternatively, the instructional material may describe one or moremethods of diagnosing HIT/HITT in a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the positive control solution of the invention or beshipped together with a container which contains the positive controlsolution. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the positive control solution be used cooperatively by therecipient.

The invention also includes a kit for use in treating a mammal afflictedwith HIT/HITT. The kit comprises a composition comprising a monoclonalantibody of the invention, or a functional element thereof, which bindsspecifically with a PF4/heparin complex. The composition can be any ofthe inventive compositions described herein. The monoclonal antibody orfunctional element thereof in the inventive composition preferentiallybinds with the PF4/heparin complex relative to the binding of themonoclonal antibody or functional element thereof with either PF4 orheparin alone. The monoclonal antibody or functional element thereof ispresent in the composition in an amount effective to competitivelyinhibit the specific binding of a polyclonal antibody in the mammal tothe PF4/heparin complex.

The kit also includes an instructional material describing the use ofthe composition comprising the monoclonal antibody or functional elementthereof for the treatment of HIT/HITT in the mammal. The instructionalmaterial is as described herein, and can be used to communicate theusefulness of the composition comprising the monoclonal antibody of theinvention or functional element thereof in the kit for treating a mammalafflicted with HIT/HITT. Optionally, or alternatively, the instructionalmaterial may describe one or more methods of treating a mammal afflictedwith HIT/HITT. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains thecomposition comprising the monoclonal antibody of the invention orfunctional element thereof, or be shipped together with the containerwhich contains the composition comprising the monoclonal antibody of theinvention or the functional element thereof. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the composition beused cooperatively by the recipient.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Example 1

The experiments discussed in this Example describe the preparation andcharacterization of a murine monoclonal antibody of the invention termed“KKO”. The materials and methods used in the experiments of this Exampleare now described.

Materials

The murine myeloma cell line P3X63Ag8U.1 (P3U1) was purchased fromAmerican Type Tissue Culture Collection (Rockville, Md.). Tissue culturereagents were from Gibco BRL (Rockville, Md.). Fetal bovine serum wasfrom Hyclone (Logan, Utah). Heparin solutions used in these studies werefrom Elkins-Sinn (Cherry Hill, N.J.). Reagents purchased from AmershamPharmacia Biotech (Piscataway, N.J.) included: ECL™ chemiluminescencedetection kit, Hi Trap affinity columns, ProRPC® 15 micron HR 10/10chromatography column, 5-hydroxy [side chain-2-¹⁴C] tryptamine creatinesulfate and the Sequenase T7 DNA polymerase kit.

Immunochemicals used in the studies described below include: anti-PF4,anti-NAP-2 and anti-IL-8 raised in rabbits from Preprotech (Rocky Hill,N.J.), swine anti-rabbit (HRP conjugate) antibody from Dako Corporation(Carpinteria, Calif.) and goat anti-human Ig G,A,M from ICN (Costa Mesa,Calif.). ImmunoPure® Monoclonal Antibody Isotyping Kit and the BCAProtein Assay Reagent Kit were obtained from Pierce Co. (Rockford,Ill.). Plates (6-well, 24-well and 96-well) for tissue culture wereproducts of Becton Dickinson (Franklin Lakes, N.J.). Maxisorp microtiterplates used for the PF4/heparin ELISA were from Nunc Brand Products(Roskilde, Denmark). Colorimentric readings were measured using aMolecular Devices plate reader (Sunnyvale, Calif.).

Molecular biological reagents included: pT₇₋₇ vector, and E. Colibacterial strain BL21(DE3)pLysS from Novagen (Madison, Wis.), VENTpolymerase from New England Biolabs Inc. (Beverly, Mass.), the murinelambda FIX 129SV library from Stratagene (LaJolla, Calif.), TRIzolreagent and Superscript RT-PCR kit from Gibco/BRL, (Gaithersburg, Md.)and AmpliTaq from Perkin-Elmer (Branchburg, N.J.). MacVector (v. 6.0)software package was purchased from Oxford Molecular Group (Oxford, UK).All other chemicals and reagents were purchased from Sigma Chemical Co.(St. Louis, Mo.).

Patient Plasma Samples

Plasma was obtained from patients having a clinical diagnosis ofHIT/HITT (Sheridan et al., 1986, Blood 67:27) and from healthyvolunteers. The clinical diagnosis was confirmed using the ¹⁴C-SerotoninRelease Assay (SRA) (Arepally et al., 1995, Am. J. Clin. Pathol.104:648) and by measuring antibodies to the PF4/heparin complex usingthe ELISA method described below. Institutional approval was obtainedfor these studies.

Development of Monoclonal Antibodies to hPF4/Heparin and hPF4

Four 6-8 week old female Balb/c mice were injected intraperitoneally(IP) on Day 1 with a 50 microliter sterile solution composed of 25microliters of PBS containing recombinant hPF4 (50 micrograms), heparin(2 Units), and 25 microliters of Freund's complete adjuvant. Subsequentinjections containing 50 micrograms of hPF4 and 2 Units of heparin inPBS were administered via the IP or tail vein on Days 12, 30, 41 and 48and 62. Mice were given an intravenous boost of heparin and hPF4 on Day66, three days prior to sacrifice. Titers of anti-hPF4/heparin weremonitored using the ELISA method described below. The two miceexpressing the highest serum titers (>1:100,000) of antibodies tohPF4/heparin complex antibodies were sacrificed and their spleens wereremoved for fusion.

Development of Monoclonal Antibodies

Fusion and hybridoma selection were optimized using standard methodology(Lane et al. 1986, Methods Enzymol 121:1183). Hybridomas were culturedfor 7 days and their supernatants screened for antibodies tohPF4/heparin and hPF4 by ELISA. Wells considered positive (A₄₀₅>0.8)were weaned from HAT supplement over 7-10 days, subcloned by limitingdilution, and grown in pristane primed mice to generate ascites.Monoclonal antibodies to hPF4/heparin (KKO) and hPF4 alone (RTO) wereisolated from ascitic fluid using the Hi Trap® affinity columnsaccording to the manufacturer's instructions. Isotyping was performedusing the ImmunoPure® Monoclonal Antibody Isotyping kit according to themanufacturer's instructions.

Preparation of Human and Murine PF4, hPF4 Mutants, NAP-2, and IL-8

Recombinant wild-type human and mouse PF4, mutant hPF4, and hNAP-2 andhIL-8 were expressed in E. coli as described in Park et al., 1990, Blood75:1290. Briefly, cDNA constructs for each chemokine (see discussionbelow) were inserted in a pT7-7 vector, introduced into E. coliBL21(DE3) pLysS, and grown in Luria broth containing 100 micrograms permilliliter ampicillin. Bacteria were grown to an A₆₀₀ of 1.0 followed bya 3-hour induction at 37° C. with 1 millimolar IPTG. Bacteria were lysedand sonicated, and the chemokine was purified at room temperature byaffinity chromatography using heparin-agarose equilibrated with 50millimolar Tris HCl, 1 millimolar EDTA, pH 8, and eluted using agradient from 0.2 to 2.0 molar NaCl. Eluted proteins were furtherpurified by reverse-phase chromatography using a ProRPC FPLC column.

Protein purity was assessed using 15% (w/v) sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis followed by Coomassie bluestaining (Park et al., 1990, Blood 75:1290). Samples were also subjectedto immunoblotting after electrotransfer to polyvinylidenedifluoride(PVDF) membranes using a commercial rabbit anti-hPF4, anti-hNAP-2 oranti-hIL-8 polyclonal antibody, followed by swine anti-rabbit secondaryantibody conjugated to horse-radish peroxidase. Proteins were detectedby ECL as described by the manufacturer. Protein concentrations weredetermined by the bicinchoninic acid (BCA) assay using BSA as thestandard according to the manufacturer's instructions.

Recombinant hPF4 variants were generated using overlap PCR as previouslydescribed in Ziporen et al., 1998, Blood 92:3250. These constructs usedwild-type hPF4 cDNA as templates and VENT polymerase enzyme. Thesequence of each mutant construct was verified using the Sequenase T7DNA Polymerase Kit. Several new hPF4 constructs were generated,designated P37N, T38Q, A39V, L41V, N47D PF4, that refer to specificamino acids in the 3rd domain of PF4 that were switched individually tothose found in hNAP-2.

The cDNA corresponding to the coding region of mature murine PF4 (mPF4)was derived from sequencing an isolated murine lambda FIX 129 SV libraryclone using human PF4 cDNA as a probe. The coding primers for mPF4 wereused to amplify mPF4 cDNA from murine platelet RNA by RT-PCR. MaturemPF4 cDNA was subcloned into pT7-7 vector with an ATG added in-frame atits N-terminus. cDNAs encoding for mature NAP-2 and IL-8 were based onpreviously published sequences and isolated cDNAs (Ziporen et al., 1998,Blood 92:3250; Baggiolini et al. 1989, J. Clin. Invest. 84:1045).

ELISA Method for Detecting Antibodies to PF4/Heparin Complex

The binding of antibody to either PF4, PF4 variants, PF4 bound tovarious GAGs or other chemokines complexed to heparin, was measuredusing an ELISA-based method as previously described (Arepally et al.,1995, Am. J. Clin. Pathol. 104:648). Briefly, to screen hybridomaculture supematants, 96-well microtiter plates were coated overnight atroom temperature with 50 micoliters per well of phosphate bufferedsaline (PBS) containing PF4 (final concentration 10 micrograms permilliliter) in the presence or absence of heparin (0.2Units/milliliter). The plates were then washed three times withTBS/0.01% Tween-20, blocked with 10% fetal calf serum (FCS) (200microliters per well) in PBS for 2 hours at room temperature, and washedonce more. Culture supernatant was added (50 microliters per well) for 1hour at room temperature. Non-bound antibody was removed by washing, and50 microliters per well of alkaline-phosphatase conjugated goatanti-mouse IgG, diluted 1:1000 in 10% FCS/PBS was added for 1 hour atroom temperature. After washing, 50 microliters per well of Sigma Fast™p-nitrophenyl phosphate substrate was added, and the absorbance at 405nanometers was measured. The binding of KKO to PF4 and relatedchemokines and the binding of KKO to PF4 bound to various GAGs, wasmeasured in the same manner, except that an incubation volume of 100microliters per well was used.

Competition Assays by ELISA

The capacity of KKO to bind to wells coated with hPF4/heparin complex inthe presence of HIT sera obtained from HIT patients was assessed using aconcentration of KKO (diluted in 10% FCS/PBS) which provided 75% ofmaximal binding. The diluted KKO was added to wells in the presence ofvarying concentrations of HIT/HITT sera. The binding of KKO tohPF4/heparin complex was measured using the ELISA procedure describedabove.

Cell Associated ELISA

Cultured human umbilical vein endothelial cells (HWVECs) were preparedas described in Jaffe et al., 1973. J. Clin. Invest. 52:2745. Cells weregrown to confluence in complete media containing M199 supplemented with20% FCS, 100 micrograms per milliliter, penicillin, 100 micrograms permilliliter, streptomycin, 5 micrograms per milliliter, amphotericin B,endothelial cell growth supplement and heparin (100 micrograms permilliliter) in 75 cm² flasks. The cells were then seeded onto 96 wellmicrotiter plates (Jaffe et al., J. Clin. Invest. 52:2745) at a densityof 32,000 cells per well in the absence of heparin. After 48 hours inheparin-free medium, the cells were fixed with 0.05% glutaraldehyde andthe binding of KKO to the cells or RTO was measured by the ELISA methoddescribed above. In other experiments, the binding of KKO to CHO cellslacking xylosyl transferase (provided by. C. Esko, Univ. CA and K.Williams, Thomas Jefferson Univ.) was conducted in essentially the samemanner.

Platelet Activation by KKO

Platelet activation by KKO in the presence of PF4 and heparin wasassayed by the release of ¹⁴C-serotonin, with modification for amicrotiter well format. Briefly, citrated platelet rich plasma obtainedfrom aspirin-free healthy donors was labeled with ¹⁴C-serotonin (22.5nCi/mL PRP) for 30 minutes at 37° C., after which platelet uptake of¹⁴C-serotonin was blocked by addition of excess imipramine (1 micromolarfinal concentration). KKO or isotype control (30-320 micrograms permilliliter) in modified Tyrode's buffer (137 mM NaCl, 3 mM KCl, 0.4 mMNaH₂PO₄, 12 mM NaHCO₃, and 1 mM MgCl₂.6H₂O, pH 7.0) was pre-incubatedwith either hPF4 (10 micrograms per milliliter) alone, heparin alone(0.2 U/mL), or h-PF4 (10 micrograms per milliliter) plus heparin (0-100U/mL). Labeled platelets (75 microliters) were added in triplicate towells containing antigen/antibody-containing mixture (20 microliters)with 5 microliters heparin or buffer to yield a 100 microliters finalvolume. After a 1 hr incubation at RT, the reaction was terminated bythe addition of 100 microliters of 0.5% NaEDTA (pH, 8.5), plateletspelleted and release of ¹⁴C-serotonin measured by liquid scintillation.In other experiments, ¹⁴C-labeled platelets were incubated with theFc-gamma RIIA blocking antibody mAb IV.3 (7.2 or 72 micrograms permilliliter) for 1 hour prior to the addition of KKO (250 micrograms permilliliter), PF4 and heparin. Controls for the assay included plasmafrom HIT patients (SRA+, positive control), normal plasma, mIgG₂b(isotype control), RTO and calcium ionophore A23187.

Sequence Analysis of KKO and RTO

Total RNA was prepared from approximately 4×10⁶ hybridoma cells forclones KKO and RTO (TRIzol reagent) followed by cDNA synthesis primedwith oligo dT (Superscript Preamplification kit) following themanufacturer's instructions. Heavy and light chain immunoglobulinvariable regions were amplified using the polymerase chain reaction aspreviously described in Siegel et al., 1994, Blood 83:2334 using thefollowing framework 1 and constant region primers for murine gamma andkappa chains: heavy chain forward 5′-GAGGTGAAGCTGGTGGAG(T/A)C(T/A)GG-3′(SEQ ID NO:5), heavy chain reverse 5′-GGGGCCAGTGGATAGAC-3′ (SEQ IDNO:6), light chain forward 5′-CCAGTTCCGAGCTCCAGATGACCCAGACTCCA-3′ (SEQID NO:7), light chain reverse 5′-GTTGGTGCAGCATCAGC-3′ (SEQ ID NO:8). ThePCR products (350-400 bp) were gel purified by electroelution anddirectly sequenced using the above oligonucleotides and automatedfluorescence sequencing. Use of these “universal” variable regionframework 1 primers provided heavy and light chain sequences that beganat the eighth and ninth amino acid residues, respectively. To determinethe authentic amino acid N-terminal residues for KKO chains, putativeleader sequences were determined by searching Genbank for other murinemonoclonal antibodies with close homology to KKO. For the heavy andlight chains of KKO, sequence accession numbers AF025443 and M20830provided candidate leader sequences. A set of 5′ PCR primers beginningat the 5′ end of the leaders were synthesized(5′-ATGGGATGGAGCTATATCATCC-3′ (SEQ ID NO:9) for heavy chain;5′-ATGATGAGTCCTGCCCAGTTCC-3′ (SEQ ID NO:10) for light chain). PCRamplifications of KKO heavy and light chain cDNA were performed usingthese primers paired with the original set of constant region reverseprimers. PCR products of the appropriate size (400-450 bp) were obtainedand served as templates to provide full-length variable region KKOsequence. Immunoglobulin gene family assignments for heavy and lightchains were determined using the Kabat (Kabat et al., 1991, “Sequencesof Proteins of Immunological Interest”, 5^(th) ed. Bethesda, NationalInstitutes of Health) and Genbank databases. Alignments of the predictedamino acid sequences were performed using the MacVector softwarepackage.

Results

Isolation and Screening of Murine Monoclonal Antibodies

Seven days post-fusion, 128 of 1152 wells contained antibodies specificfor either a hPF4/heparin complex or hPF4 alone as determined by ELISAusing an A₄₀₅ greater than or equal to 0.8 as an arbitrary cut-offvalue. Cells were subcultured when their supernatants generated A₄₀₅ratios to hPF4/heparin complex versus hPF4 alone of greater than 1.5.Cell populations showing the greatest relative specificity for thehPF4/heparin complex versus hPF4 alone, and those with high reactivityto hPF4 alone, underwent three additional rounds of subcloning. Twomonoclonal antibodies, an IgG_(2b)κ antibody specific for thehPF4/heparin complex designated KKO and an IgG_(2b)κ antibody specificfor hPF4 alone designated RTO, were ultimately isolated and subjected tofurther characterization. KKO exhibited an A₄₀₅ ratio of greater than 30to hPF4/heparin complex versus hPF4 alone, whereas RTO demonstrated anA₄₀₅ ratio of less than or equal to 1. These monoclonal antibodies werepurified from ascites and were used for all subsequent studies.

Specificity of KKO and RTO for PF4/Heparin Complexes

KKO bound to hPF4/heparin complexes in a dose-dependent manner, as shownin FIG. 1A. Half-maximal binding was exhibited at an antibodyconcentration of 0.036 micrograms per milliliter. The relativespecificity of KKO was determined by adding various dilutions of KKO towells coated with hPF4/heparin complex or hPF4 alone. Specificity forthe complex was evident at all antibody concentrations tested from 0.007to 36 micrograms per milliliter (FIG. 1A). At concentrations less than0.14 micrograms per milliliter, the ratio of binding of KKO toPF4/heparin complex vs. binding to PF4 alone as determined by A₄₀₅ wasgreater than 400. No binding of KKO to PF4 alone was evident atconcentrations less than or equal to 0.072 micrograms per milliliter.KKO did not exhibit binding to immobilized heparin at any concentrationtested from 0.007-36 micrograms per milliliter. Whereas KKO demonstratedheparin-dependent binding to hPF4, binding of RTO to hPF4 was unaffectedby the presence of heparin at all concentrations of antibody tested(FIG. 1B). Naturally-occurring antibodies in patients with HIT recognizehPF4/heparin complexes formed over a narrow range of molar ratiosbetween the reactants (Kelton et al., 1994, Blood 83:3232).

To determine if KKO exhibited similar characteristics, antibody bindingto HPF4/heparin complex was measured at a fixed concentration of hPF4(10 micrograms per milliliter) and varying concentrations of heparin(0.01-50 Units/mL). Optimal binding of KKO occurred at a molar ratio ofhPF4 to heparin of 3:1. Reduced KKO binding to hPF4/heparin complex wasobserved in the presence of higher and lower concentrations of heparin(FIG. 1C).

Binding of KKO to Human PF4 in Complex with GAGs

Another feature of naturally-occurring HIT antibodies is theircross-reactivity with complexes composed of PF4 and other sulfatedglycosaminoglycans (GAGs) (Greinacher et al., 1992, Thromb. Haemost.67:545; Greinacher et al., 1995, Thromb. Haemost. 74:886). As shown inFIG. 2, KKO bound to complexes formed between hPF4 (10 micrograms permilliliter) and 0-500 micrograms per milliliter chondroitin sulfate A,chondroitin sulfate B or dermatan sulfate, chondroitin sulfate C,heparan sulfate and dextran sulfate (M_(r) 8000), in a pattern ofbinding similar to that reported previously for HIT antibodies(Greinacher et al., 1994, Thromb. Haemost. 71:247).

Cell-Reactivity of KKO

Similar to HIT-IgG (Visentin et al., 1994, J. Clin. Invest. 93:81), KKObound to cultured endothelial cells (FIG. 3) and CHO cells in thepresence of exogenous hPF4, but not to CHO cells lacking heparansulfate- or chondroitin sulfate-containing proteoglycans under the saneconditions. Binding of KKO to HUVECs was inhibited by heparin atconcentrations (0.2 Units/mL) known to dissociate PF4 from the cellsurface. In comparison to KKO, equimolar concentrations of RTO boundone-third as well to HUVEC in the presence of either PF4 or PF4 andheparin, consistent with ELISA data shown in FIGS. 1A and 1B. Whereasbinding of KKO to PF4 was enhanced in the presence of heparin (FIG. 1A),binding of RTO was diminished (FIG. 1B), suggesting that RTO recognizesan epitope on PF4 that is masked or altered by heparin or heparin-likemolecules upon formation of the PF4/heparin complex.

Platelet Activation by KKO

HIT antibodies (Kelton et al., 1988, Blood 72:925), and immune complexescontaining murine IgG₂ antibodies activate human platelets through aprocess which requires Fc-gamma RIIA (Hewlett et al., 1994, Adv. Immunol57:1; De Reys et al., 1993, Blood 81:1792). In order to determinewhether KKO activates platelets through a similar pathway,¹⁴C-serotonin-labeled platelets were incubated with KKO (atconcentrations ranging from 30 to 320 micrograms per milliliter) in thepresence of either hPF4 alone, heparin alone, or hPF4/heparin complex.KKO at concentrations of 80 and 160 micrograms per milliliter)stimulated ¹⁴C-serotonin release in a heparin-dependent manner as shownin FIG. 4 when pre-incubated with 10 micrograms per milliliter of hPF4.Somewhat higher concentrations of antibody (KKO greater than 180micrograms per milliliter) were required to initiate serotonin releasewhen the antibody was pre-incubated with 10 micrograms per milliliter ofhPF4 complexed to heparin (1 Unit/mL). Neither KKO nor the isotypecontrol activated platelets in the presence of buffer alone or heparinalone. The release of ¹⁴C-serotonin induced by KKO was almost completelyinhibited by the FcγRIIA-specific mAb IV.3 (less than 5% release at 0.5,1, and 5 Units/mL heparin). RTO did not exhibit the ability to effectserotonin release in the presence or absence of hPF4 alone orhPF4/heparin complex at all concentrations tested (from 31-250micrograms per milliliter).

Epitope Specificity of KKO

It has been reported that a subset of HIT/HITT antibodies require anepitope in the 3rd domain of hPF4 in order to bind hPF4 in the presenceof heparin. This region was defined using chimeras between hPF4 and thestructurally related chemokine, NAP-2, which is not recognized by HITantibodies (Ziporen et al., 1998, Blood 92:3250). In order toinvestigate both the specificity of this subset of HIT antibodies ingreater detail and the involvement of this domain in the binding of KKO,single amino acid substitutions were introduced into the 3rd domain ofPF4, between Cys36 and Cys52. Each PF4 variant was incubated with anoptimal concentration of heparin (Ziporen et al., 1998, Blood 92:3250)and the binding of KKO with sera obtained from 23 HIT patients wasassessed.

The binding of HIT antibodies to heparin complexed with the PF4 variantsP37N, T38Q, A39V, L41 V, and N47D was moderately impaired, compared withwild type PF4, whereas binding to NAP-2 was minimal. Binding of HITantibodies to the hPF4 variants exhibited greater variability thanbinding to wild type hPF4, suggesting differences in the proportion ofantibodies in individual sera sensitive to changes in the third domainof hPF4. HIT sera could be divided arbitrarily into subgroups exhibitingmarked (A₄₀₅ of less than 1.0), intermediate (A₄₀₅ greater than or equalto 1.0 to less than 2.5), or little (A₄₀₅ of greater than 2.5),sensitivity to the P37N mutation. The binding of HIT antibodies to PF4variants having mutations in the 3rd domain (T38Q, A39V, L41V, and N47D)generally displayed the same pattern of reactivity (i.e., seracontaining antibodies which recognized P37N also recognized T38Q, etc.).These data suggest that there are at least two antigenic sitesrecognized by antibodies in sera obtained from patients with HIT. Serafrom most HIT patients contain antibodies which recognize both sites,but a subgroup are comprised of antibodies which predominantly recognizean antigenic site (or sites) involving the 3rd domain of hPF4.

The antigenic site recognized by KKO was characterized next. It has beenreported previously that greater than 95% of HIT antibodies do notrecognize IL-8 or NAP-2 complexed to heparin (Ziporen et al., 1998,Blood 92:3250; Amiral et al., 1996, Blood 88:410), although IL8 andNAP-2 share about 30% and about 60% amino acid sequence homology,respectively, with hPF4. KKO shares this characteristic and does notbind to either chemokine when complexed to heparin, as shown in FIG. 5A.Secondly, none of the 23 HIT sera tested or KKO reacted with murine PF4complexed to heparin (FIG. 5A), in spite of mPF4 displaying, about 80%amino acid sequence identity with its human homologue. Thirdly, KKObound strongly to the PF4 variants P37N, T38Q, A39V and L41V in thepresence of heparin (FIG. 5A), similar to the behavior of the sub-groupof HIT sera characterized as “insensitive” to mutations in the thirddomain.

In light of these findings, cross-competition experiments were thenperformed to determine whether KKO and this subset of HIT antibodiesrecognized an overlapping site in PF4. To do so, the binding of KKO tohPF4/heparin complex was measured in the presence of increasing amountsof four 3rd-domain “insensitive” HIT sera and five “sensitive” sera(FIG. 5B). Three of four “insensitive” serum samples tested inhibitedthe binding of KKO to hPF4/heparin complex by about 50%. In contrast,none of the 5 sera designated “sensitive” inhibited binding of KKO to asimilar extent. These data are consistent with the pattern of KKObinding to the 3rd domain mutants shown in FIG. 5A and suggest that KKOrecognizes an epitope of the hPF4/heparin complex which overlaps withone recognized by a subset of HIT antibodies.

Sequence Analysis of KKO and RTO

The predicted amino acid sequence of KKO, a PF4/heparin complex-specificmurine monoclonal antibody, and RTO, a non-heparin dependent anti-PF4murine monoclonal antibody were compared. Results are shown in FIGS. 6Aand 6B. Sequence analysis revealed the use of very disparate V_(H)families and J_(H)-gene segments and V_(L) families and J_(L) genesegments for KKO (SEQ ID NOs:11 and 13) and RTO (SEQ ID NOs: 12 and 14)heavy and light chains, respectively. Although one cannot rule outsimilarities in the idiotopes expressed by two antibodies based on theirprimary heavy and light chain sequences, it is clear that KKO and RTOare not genetically (or clonally) related, nor do they bear any obviouspredicted structural homology to each other.

Discussion

A murine monoclonal antibody termed KKO was generated which bindsspecifically with a PF4/heparin complex and which binds preferentiallywith the PF4/heparin complex relative to its binding with either PF4 orheparin alone. KKO shares important serologic and functional propertieswith naturally-occurring anti-PF4/heparin complex antibodies obtainedfrom sera of patients with heparin-induced thrombocytopenia/thrombosis(HITT). KKO recognizes PF4 in complex with heparin over a narrow rangeof molar ratios approaching 1:1, similar to the behavior ofnaturally-occurring HIT antibodies (Amiral et al., 1992. Thromb. Haemost68:95). KKO also binds specifically with complexes between PF4 and otherglycosaminoglycans (GAGs), but not with heparin itself, or with heparincomplexed with either mPF4, NAP-2, or IL-8. These features are sharedwith greater than 95% of HIT antibodies. KKO binds to cells whichexpress GAGs (such as endothelial cells) only when PF4 is providedexogenously (Herbert et al., 1998, Thromb. Haemost 80:326; Visentin etal., 1994, J. Clin. Invest. 93:81; Tannenbaum et al., 1986, J. Immunol137:1532). It does not bind to cells which lack GAGs and which aretherefore unable to form the requisite antigenic complex. KKO alsoactivates human platelets through a heparin- and PF4-dependent mechanismthat is mediated through Fc-gamma RIIA (Kelton et al., 1988, Blood72:925).

The binding of KKO to the PF4/heparin complex exceeded binding of KKO toPF4 alone by greater than 400 fold under optimal binding conditions.However, the finding that KKO binds to PF4 at high concentrations of KKOis consistent with the hypothesis that heparin and other GAGs induce aconformational change in the protein which generates neoepitopes withinthe PF4 molecule which are responsible for antibody formation (Ziporenet al., 1998, Blood 92:3250; Horsewood et al., 1996, Br. J. Haematol.95:161; Mayo et al., 1995, Biochem J. 312:357). The fact that miceinjected with heparin and PF4 generate PF4/heparin complex-specificantibodies indicated the immunogenicity of these putative neoepitopesand may have implications for the mechanism by which antibody formationis stimulated in humans.

The results of several studies have indicated that naturally-occurringantibodies specific for the PF4/heparin complex are polyspecific (Suh etal., 1998, Blood 91:916; Ziporen et al., 1998, Blood 92:3250). KKOcompetes with a subset of these antibodies for binding to thePF4/heparin complex. A region has previously been identified in the 3rddomain of PF4 which is required for recognition by a subset of HIT/HITTantibodies (Ziporen et al., 1998, Blood 92:3250). Additional mutationsin this region affirm its contribution to the immunodominant epitoperecognized by some, but not other, naturally-occurring antibodies. Theresults of direct binding studies using these variant PF4 molecules, aswell as competition studies using HIT/HITT sera subdivided by theircapacity to recognize these variants, suggest that the epitope for KKOlies outside of this third domain. A comparison of the amino acidsequences of KKO, a PF4/heparin complex-specific antibody, with RTO, aPF4-specific antibody, fails to suggest any obvious genetic relatednessor structural similarity between the combining regions of theseantibodies or any relationship between their binding sites on PF4.

The propensity of patients with HIT to develop thrombosis has beenattributed to antibody-mediated platelet activation in vivo (Kelton etal., 1988, Blood 72:925; Fratantoni et al., 1975, Blood 45:395).Interestingly, the concentrations of KKO required to activate platelets(i.e., at least 80 micrograms per milliliter) greatly exceed the minimalamount of KKO required to detect the PF4/heparin complex by ELISA (0.036micrograms per milliliter). In addition to reflecting the difference insensitivity of these two assays (Bauer et al., 1997, Circulation95:1242; Visentin et al., 1996, J. Lab. Clin. Med. 128:376), thisfinding might indicate a threshold of Fc receptor occupancy that must beexceeded in order for KKO to initiate platelet activation.Naturally-occurring differences, either in the expression of FcγRIIA(Rosenfeld et al., 1987, J. Immunol 138:2869: McCrae et al., 1990, J.Immunol. 144:3920), the extent to which receptor expression isupregulated when platelets are activated (McCrae et al., 1990, J.Immunol. 144:3920; Chong et al., 1993, Blood 81:988), the presence ofhigher titers of IgG antibodies in patients with thrombosis (Suh et al.,1997, Am. J. Hematol. 54:196), or the poorly understood role of theplatelet Fc-gamma RIIA-H/R¹³¹ polymorphism, may each contribute to thepropensity of some patients with sensitization to the PF4/heparincomplex to develop overt disease. Platelet activation by KKO may belimited both by the number and conformation of the PF4/heparin complexeswhich can bind to the platelet surface, and by the affinity of murineIgG_(2b) for human Fc-gamma RIIA.

Although the relationship between plasma PF4/heparin complex-specificantibodies and HIT is well established, there is no formal proof thatthese antibodies, or a subset of these antibodies, actually causethrombocytopenia or thrombosis. It has been reported previously thatmice immunized with HIT-IgG develop murine antibodies to humanPF4/heparin complexes and PF4-dependent endothelial cell-reactiveantibodies as part of the idiotype/anti-idiotype network (Blank et al.,1997, Clin. Exp. Immunol. 108:333). Immunized mice developedthrombocytopenia when exposed to heparin. However, as these mice likelydeveloped antibodies with additional specificities as a result ofepitope spread, the contribution of PF4/heparin complex-specificantibodies per se to the development of thrombocytopenia or thrombosisremains unproven. PF4/heparin complex-specific monoclonal antibodiessuch as KKO facilitate efforts to identify the pathophysiological roleof anti-PF4/heparin complex antibodies in vivo. It is also anticipatedthat KKO and related antibodies will be useful to elucidate thephysiologic role of PF4 in hemostasis, serve as a reagent standard inmethods for diagnosing HIT/HITT, and may provide a platform from whichto identify therapeutic alternatives for patients with HIT/HITT.

Example 2

The experiments described in this Example provide evidence that KKO, amurine monoclonal antibody of the invention, induces the secretion ofIL-8 by monocytes in the presence of PF4, but not in the presence ofheparin alone or a PF4/heparin complex. The cytokine IL-8 is often usedas a surrogate marker for activated monocytes and monocyte-like cells.

Using the monocytic cell line U937, various treatments with stimulantswere tested in the cells (no stimulant, TNF-alpha, KKO, an isotypecontrol antibody TRA-2b, sera from a patient with HIT, and sera from acontrol patient) in the presence of either PF4 (10 micrograms permilliliter), heparin (1 Unit per milliliter) or the PF4/heparin complex(10 micrograms per milliliter and 1 Unit per milliliter, respectively).The cells were cultured for 24 hours with the various stimulants, andthe secretion of IL-8 was measured using a commercially available assay(R&D Systems, Minneapolis, Minn.).

As depicted in FIG. 8, a significant amount of IL-8 secretion by thecells was observed, which was increased in the presence of PF4 with KKOrelative to the isotype control antibody, and with HIT sera as comparedto control sera. Without wishing to be bound by any particular theory,it is suspected that KKO binds with PF4 complexed to cell surfaceglycosaminoglycans, which leads to the activation of monocyte-likecells. These results are similar to findings which indicate increasedIL-8 secretion by HIT sera relative to control sera.

These data provide evidence that the murine monoclonal antibody of theinvention KKO participates in the pathogenesis of HIT/HITT. Since KKObinds and activates monocytes, tissue factor expression may be increasedby KKO, which promotes the hypercoagulable state of HIT/HITT. Theactivation of monocytes by HIT/HITT antibodies can be used as adiagnostic marker, and the blocking of monocyte activation by amonoclonal antibody of the invention such as KKO or a derivative thereofcan be used therapeutically to halt the development of thrombosis.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A Fab antibody capable of binding specifically with a platelet factor4 (PF4)/heparin complex, wherein said antibody preferentially binds withsaid PF4/heparin complex relative to the binding of said antibody witheither PF4 or heparin alone, wherein the Fab antibody comprises anantibody-binding fragment comprising the amino acid sequence of SEQ IDNO: 11, and wherein the Fab antibody further comprises the amino acidsequence of SEQ ID NO:13.
 2. A kit for diagnosing HIT/HITT in a mammal,said kit comprising: (a) a positive control solution comprising amonoclonal antibody or antigen-binding fragment thereof which bindsspecifically with a platelet factor 4 (PF4)/heparin complex, whereinsaid monoclonal antibody or antigen-binding fragment thereofpreferentially binds with said PF4/heparin complex relative to thebinding of said monoclonal antibody or antigen-binding fragment thereofwith either PF4 or heparin alone, and wherein the monoclonal antibody orantigen-binding fragment thereof comprises the amino acid sequence ofSEQ ID NO: 11 and the amino acid sequence of SEQ ID NO:13; and (b) aninstructional material describing the use of said positive controlsolution for diagnosing HIT/HITT in a mammal.
 3. The kit of claim 2wherein the monoclonal antibody is produced by hybridoma KKO depositedunder ATCC Accession No: PTA-6133.
 4. A kit for diagnosing HIT/HITT in amammal, said kit comprising: (a) a positive control solution comprisinga monoclonal antibody or an antigen-binding fragment thereof which bindsspecifically with a platelet factor 4 (PF4)/heparin complex, whereinsaid monoclonal antibody or antigen-binding fragment thereofpreferentially binds with said PF4/heparin complex relative to thebinding of said monoclonal antibody or antigen-binding fragment thereofwith either PF4 or heparin alone, and wherein the monoclonal antibody isproduced by hybridoma KKO deposited under ATCC Accession No: PTA-6133;and (b) an instructional material describing the use of said positivecontrol solution for diagnosing HIT/HITT in a mammal.